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Molecular Studies of the Pathogenic Free-living
Amoeba, Acanthamoeba
Thesis submitted for the degree of
Doctor of Philosophy
at the University of Leicester
by
Kimberley Amy Durham B.Sc. M.Sc.
Department of Infection, Immunity & Inflammation
University of Leicester
April 2012
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Abstract
Molecular Studies of the Pathogenic Free-living Amoeba, Acanthamoeba
By Kimberley A. Durham
Ubiquitous amoebae from the genus Acanthamoeba are associated with two
main serious infections: The more common eye disease acanthamoeba keratitis
(AK), which can result in blindness, and the rare and often fatal disease affecting
the central nervous system, granulomatous amoebic encephalitis (GAE).
The traditional morphological taxonomic system for Acanthamoeba is based
on cyst size and shape, and divides the amoebae into three groups (I, II and III).
Since the discovery that cyst shape can be modified by culture conditions, the
classic system has become largely redundant. A more robust system has been
developed, based on the nucleotide sequence of the 18S rRNA (Rns) gene. It types
Acanthamoeba into 15 T-groups, with most species including environmental and
clinical clumped into three groups T3, T4 and T11, with little resolution between
them. Although speciation does not help cure patients directly, it can provide
valuable information regarding disease epidemiology and ultimately benefit patient
prognosis.
Here a system to better resolve strains has been developed, using the
mitochondrial cytochrome oxidase subunit 1 and 2 (cox1/2) gene sequence. When
used in conjunction with the T-group system, resolution between strains including
those with a T3, T4 or T11 genotype is obtained. Additionally the combined
approach identified a mixed infection in a patient suffering with AK, and the
occurrence of Acanthamoeba strains with multiple alleles of 18S and cox1/2 genes.
The combined use of both genotyping systems was used to investigate an
unprecedented outbreak of GAE within a Swedish hospital. Results confirmed
Acanthamoeba had infected several immunocompromised paediatrics from a single
ICU, and the source was from within the unit’s water system. In vitro assays were
used to test the strains pathogenic abilities and sensitivities to antimicrobial
compounds, identifying if they are more virulent than typical strains of
Acanthamoeba.
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Acknowledgements
This work was supported by sponsorship funding from Bausch and Lomb. I
also owe thanks to most of the members of the Department of Infection, Immunity
and Inflammation for their assistance throughout my research, in particular Dr
Wayne Heaselgrave, Dr James Lonnen, Niran Patel and Professor Peter Andrew.
I would like to give a special thanks to my family for their continued support
without which this write up would not have possible.
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Table of contents
1 INTRODUCTION ............................................................................................ 1
1.1 Free-living amoebae ................................................................................... 1
1.2 Acanthamoeba ............................................................................................ 5
1.3 Acanthamoeba ecology ............................................................................... 7
1.4 Acanthamoeba cultivation ........................................................................ 10
1.5 Acanthamoeba epidemiology .................................................................... 12
1.6 Granulomatous amoebic encephalitis (GAE) ........................................... 15
1.7 Acanthamoeba keratitis (AK) .................................................................... 19
1.8 Taxonomy and Classification.................................................................... 24
1.9 Molecular biology of Acanthamoeba ........................................................ 28
1.10 Aims .......................................................................................................... 33
2 DNA TYPING OF ACANTHAMOEBA SP. ................................................. 34
2.1 Introduction ..................................................................................................... 34
2.1.1 Aims .................................................................................................. 37
2.2 Materials and Methods ................................................................................... 38
2.2.1 Chemicals .................................................................................................. 38
2.2.2 Organisms ................................................................................................. 38
2.2.3 Monoxenic culture of Acanthamoeba ....................................................... 40
2.2.3.1 Preparation of E. coli food source stock ........................................... 41
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2.2.4 Axenic culture of Acanthamoeba .............................................................. 42
2.2.5 Cryopreservation of Acanthamoeba ......................................................... 43
2.2.5.1 Cryopreservation of bacteria ............................................................. 45
2.2.6 Acanthamoeba DNA isolation .................................................................. 45
2.2.6.1 DNA extraction from Acanthamoeba in monoxenic culture ............ 46
2.2.7 Agarose gel electrophoresis ...................................................................... 47
2.2.8 Primer design ............................................................................................ 48
2.2.9 PCR amplification of DNA ....................................................................... 48
2.2.10 Purification of PCR products .............................................................. 49
2.2.10.1 PEG purification of single bands ...................................................... 50
2.2.10.1.i Micron®–PCR purification of single bands 50
2.2.10.2 Target DNA purification from a multiple band PCR ....................... 51
2.2.11 Ligation of DNA into cloning vectors .................................................. 52
2.2.12 Production of ultra competent E. coli cells ......................................... 53
2.2.13 Heat shock transformation of ultra competent E. coli ......................... 54
2.2.14 Restriction enzyme digestion ............................................................... 55
2.2.15 Plasmid purification ........................................................................... 58
2.2.15.1 Silica method of plasmid purification for screening ......................... 58
2.2.15.2 Comprehensive plasmid purification for sequencing ....................... 60
2.2.15.3 QIAGEN® plasmid maxi kit ............................................................ 61
2.2.16 Sequencing ........................................................................................... 61
2.2.17 Sequence analysis ................................................................................ 62
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2.3 Results .............................................................................................................. 63
2.3.1 18S genotyping of strains .......................................................................... 63
2.3.2 Typing strains with cox1/2 gene ............................................................... 69
2.3.3 Introns, multiple alleles and mixed infections .......................................... 77
2.3.4 Concatenated sequence data .................................................................... 78
2.4 Discussion ........................................................................................................ 84
2.4.1 Genotyping Acanthamoeba sp. with 18S sequences ................................. 84
2.4.2 Genotyping based on cox1/2 sequences .................................................... 87
2.4.3 Typing with concatenated gene sequences ............................................... 88
2.4.4 Multiple alleles and mixed infections using 18S and cox1/2 .................... 89
2.4.5 Conclusion ................................................................................................ 91
3 SWEDISH GAE STUDY ................................................................................ 93
3.1 Introduction ..................................................................................................... 93
3.1.1 Aims .................................................................................................. 95
3.2 Materials and Methods ................................................................................... 96
3.2.1 Establishing amoebae cultures ................................................................. 96
3.2.2 Amoebae DNA extraction ......................................................................... 97
3.2.2.i Direct DNA extraction from CSF 97
3.2.3 DNA manipulation .................................................................................... 98
3.2.4 Temperature tolerance assay .................................................................... 98
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3.2.5 Osmotolerance assay ................................................................................ 99
3.2.6 Protease secretion ..................................................................................... 99
3.2.7 Complement fixing potential ................................................................... 100
3.2.8 Cytopathogenic potential ........................................................................ 101
3.2.9 Antimicrobial sensitivity assays .............................................................. 102
3.2.9.1 Trophozoites ................................................................................... 102
3.2.9.2 Cysts ................................................................................................ 103
3.3 Results ............................................................................................................ 106
3.3.1 Culture and morphological analysis....................................................... 106
3.3.1.1 Patient one ....................................................................................... 106
3.3.1.2 Patient two ...................................................................................... 106
3.3.1.3 Patient three .................................................................................... 107
3.3.1.4 Hospital water samples ................................................................... 107
3.3.2 Molecular analysis .................................................................................. 109
3.3.2.1 18S phylogenetic analysis ............................................................... 109
3.3.2.2 Cox1/2 phylogenetic analysis ......................................................... 113
3.3.3 Pathogenicity assays ............................................................................... 115
3.3.3.1 Amoebicidal activity of human serum ............................................ 115
3.3.3.2 Tolerance of increased temperature and osmolarity ....................... 116
3.3.3.3 Protease secretion assays ................................................................ 116
3.3.3.4 Cytopathogenicity of Acanthamoeba .............................................. 117
3.3.4 Drug sensitivity assays ............................................................................ 120
3.4 Discussion ...................................................................................................... 124
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3.4.1 Molecular ................................................................................................ 124
3.4.2 Pathogenicity .......................................................................................... 127
3.4.3 Antimicrobial sensitivity ......................................................................... 131
3.4.1 Conclusion .............................................................................................. 136
4 REFERENCES .............................................................................................. 138
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List of Figures
Figure 1. Neighbour joining distance tree based on partial 18S rDNA sequences of
Acanthamoeba spp. The tree is unrooted and obtained by Kimura two-parameters
correction for multiple substitutions using MEGA (5.05). The tree is based on
reference bp from 1,175 to 1,379. The scale bar represents the corrected number of
nucleotide substitutions per base using Kimura method. Designated T-groups are
shown. ....................................................................................................................... 67
Figure 2. Maximum parsimony distance tree based on partial 18S rDNA sequences
of Acanthamoeba spp. The tree is unrooted and obtained by Kimura two-parameters
correction for multiple substitutions using MEGA (5.05). The tree is based on
reference bp from 1,175 to 1,379. Designated T-groups are shown. ........................ 69
Figure 3. Maximum parsimony tree based on partial mt cox1/2 sequences of
Acanthamoeba spp, with cox1/2 groups (A-H) and T-groups (T2-T11) included. The
tree is unrooted and obtained by Kimura two-parameters correction for multiple
substitutions using MEGA (5.05). The tree is based on reference bp 8,002 to 8,566.
Bootstrap values have been included, based on 1,000 bootstrap values, and are
placed at the nodes they apply to. ............................................................................. 74
Figure 4. Neighbour-joining distance tree based on partial mt cox1/2 sequences of
Acanthamoeba spp with cox1/2 groups (A-H) and T-groups (T2-T11) included. The
tree is unrooted and obtained by Kimura two-parameters correction for multiple
substitutions using MEGA (5.05). The tree is based on reference bp 8,002 to 8,566.
The scale bar represents the corrected number of nucleotide substitutions per base
using Kimura method. ............................................................................................... 76
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Figure 5. Maximum parsimony distance tree based on concatenated partial mt
cox1/2 with 18S sequences of Acanthamoeba spp with cox1/2 groups (A-H) and T-
groups (T2-T11) included. The tree is unrooted and obtained by Kimura two-
parameters correction for multiple substitutions using MEGA (5.05). The tree is
based on reference bp 8,002 to 8,566. ...................................................................... 81
Figure 6. Neighbour-joining distance tree based on concatenated partial mt cox1/2
with 18S sequences of Acanthamoeba spp with cox1/2 groups (A-H) and T-groups
(T2-T11) included. The tree is unrooted and obtained by Kimura two-parameters
correction for multiple substitutions using MEGA (5.05). The tree is based on
reference bp 8,002 to 8,566. The scale bar represents the corrected number of
nucleotide substitutions per base using Kimura method. ......................................... 83
Figure 7. Maximum parsimony distance tree based on partial 18S rDNA sequences
of Swedish hospital FLA with comparison species. The tree is unrooted and
obtained by Kimura two-parameters correction for multiple substitutions using
MEGA (5.05). Analysis is based on reference bp from 1,175 to 1,379. Designated
T-groups and strain origins are shown. Bootstrap values have been included, based
on 1,000 bootstrap values, and placed at the corresponding nodes. ....................... 112
Figure 8. Maximum parsimony tree based on partial mitochondrial cox1/2
sequences of Acanthamoeba spp. The tree is unrooted and obtained by Kimura two-
parameters correction for multiple substitutions using MEGA (5.05). The tree is
based on reference bp 8,002 to 8,566. Designated T-groups and origin of strains are
shown. Bootstrap values have been included, based on 1,000 bootstrap values, and
placed at the corresponding nodes. ......................................................................... 114
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Figure 9. Maximum parsimony tree based on partial mitochondrial cox1/2
sequences of Acanthamoeba spp. The tree is unrooted and obtained by Kimura two-
parameters correction for multiple substitutions using MEGA (5.05). The tree is
based on reference bp 8,002 to 8,566. Designated T-groups and origin of strains are
shown. Bootstrap values have been included, based on 1,000 bootstrap values, and
placed at the corresponding nodes. The scale bar represents the corrected number of
nucleotide substitutions per base using Kimura method. ....................................... 115
Figure 10. Activity of Normal Human Serum against Acanthamoeba trophozoites.
A, effect of complement from NHS on Acanthamoeba trophozoites; B, control,
Acanthamoeba incubated with heat-inactivated NHS; C, pellet formed in the round
bottomed well after lysis of Acanthamoeba trophozoites. ...................................... 115
Figure 11. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-
PAGE) containing gelatine, to observe the presence of extracellular proteases in
Acanthamoeba culture medium (ACM). Lane 1, ICU pool; 2, Shower room 3; 3,
Shower room 15; 4, A. polyphaga Ros; 5, Patient 3; 6, Patient 2; 7, Left blank; 8,
negative control, containing sterile culture media. ................................................. 117
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List of Tables
Table 1. Morphologically designated Acanthamoeba species assigned to T-groups
based on phylogenetic analysis of 18s sequence variations. .................................... 26
Table 2. Microorganisms used for these studies. ...................................................... 38
Table 3. Primers (5' to 3') used for gene sequence typing of Acanthamoeba spp.
SSU 18S rDNA and cox1/2, with corresponding PCR parameters. ......................... 56
Table 4. Estimates of evolutionary divergence over 18S sequence pairs between T-
group genotypes. The number of base substitutions per site from between sequences
are shown. Analyses were conducted using the Maximum Composite Likelihood
model (Tamura et al., 2004). The analysis involved 40 nucleotide sequences. All
positions containing gaps and missing data were eliminated. There were a total of
336 positions in the final dataset. Evolutionary analyses were conducted in MEGA5
(Tamura et al., 2011) in press. .................................................................................. 64
Table 5. Estimates of evolutionary divergence over cox1/2 sequence pairs between
groups (A-G). The number of base substitutions per site from between sequences
are shown. Analyses were conducted using the Maximum Composite Likelihood
model (Tamura et al., 2004). The analysis involved 40 nucleotide sequences. All
positions containing gaps and missing data were eliminated. There were a total of
336 positions in the final dataset. Evolutionary analyses were conducted in MEGA5
(Tamura et al., 2011) in press. .................................................................................. 71
Table 6. Pairwise distance values between cox1/2 sequences of repeat isolates from
Patient three. The number of base substitutions per site from between sequences are
shown. Analyses were conducted using the Maximum Composite Likelihood model
(Tamura et al., 2004). The analysis involved 40 nucleotide sequences. All positions
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containing gaps and missing data were eliminated. There were a total of 336
positions in the final dataset. Evolutionary analyses were conducted in MEGA5
(Tamura et al., 2011) in press. .................................................................................. 78
Table 7. FLA isolated from clinical and hospital samples collected in a Swedish
hospital, with morphological identification of genus groups. ................................ 108
Table 8. Pathogenicity characteristics of Acanthamoeba sp. collected from an
outbreak of GAE with a single ward of a Swedish hospital, and compared with an
AK isolate and a non-pathogen. .............................................................................. 119
Table 9. In vitro sensitivities of five antimicrobial compounds against several strains
of Acanthamoeba trophozoites and cysts. ............................................................... 122
Table 10. Acanthamoeba in vitro Minimum Cysticidal Concentrations (MCC),
against five antimicrobial compounds. ................................................................... 122
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Abbreviations
AK Acanthamoeba keratitis
APS Ammonium persulfate
ARB Amoeba-resistant bacteria
ATCC American Type Culture Collection
CCAP Culture Collection of Algae and Protozoa
CFU Colony forming units
CNS Central nervous system
CSF Cerebrospinal fluid
CT Computerised tomography
DH5α Escherichia coli (ATTC 53868)
DMEM Dulbecco’s Modified Eagle Medium
DMSO Dimethyl sulfoxide
dNTP Deoxynucleotides
dPBS Dulbecco’s Phosphate buffered saline
EDTA Ethylenediaminetetraacetic acid
EtOH Ethanol
FBS Foetal bovine serum
FLA Free-living amoeba
GMS Gomori methamine silver
HCl Hydrochloric acid
IIF Indirect immunofluorescence
IPTG Isopropyl-β-D-thiogalactoside
KH2PO4 Potassium dihydrogen orthophosphate
LB Luria-Bertani
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LSHTM London School of Hygiene & Tropical Medicine
LSU Large subunit
MgCl2 Magnesium chloride
MP Maximum parsimony
MRI Magnetic resonance imagery
MRSA Methicillin-resistant Staphylococcus aureus
NaCl Sodium chloride
NaOH Sodium hydroxide
NHS Normal human serum
nH2O Nanopure water
NJ Neighbour-joining
(NH4)2SO4 Ammonium sulphate
NNA Non-nutrient agar
PAGE Polyacrylamide gel
PAM Primary amoebic meningoencephalitis
PAS Periodic acid-Schiff
PCR Polymerase chain reaction
PEG Polyethylene glycol
PNACL Protein Nucleic Acid Chemistry Laboratory
RE Restriction enzymes
Rnl The large subunit rRNA gene
Rns The gene encoding for 18S
SDS Sodium dodecyl sulphate
SOB Super optimal broth
SOC SOB with catabolite repression (added glucose)
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SSU Small subunit
TEM Transmission electron microscopy
TEMED Tetramethylethylenediamine
X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactoside
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Suppliers
ABGene
The Amoebae
Laboratory
Applied Biosystems
Arch U.K. Biocides
ATCC
Barloworld Scientific
Ltd
Bausch & Lomb
Thermo Scientific (ABGene), ABgene House, Blenheim
Road, Epsom, KT19 9AP, U.K.
Department of Infection, Immunity and Inflammation,
Maurice Shock Medical Sciences Building, University
Road, Leicester, LE1 9HN.
Applied Biosystems, Lingley House, 120 Birchwood
Boulevard, Warrington, WA3 7QH, U.K.
Arch U.K. Biocides Ltd, Wheldon Road, Castleford,
West Yorkshire, WF10 2JT, U.K.
American Type Culture Collection (ATCC), LGC
Standards, Queens Road, Teddington, Middlesex, TW11
0LY, U.K.
Barloworld Scientific Ltd, Stone, Staffordshire, ST15
0SA
Bausch & Lomb U.K. Ltd, 106 London Road, Kingston
upon Thames, Surrey, KT2 6TN, U.K.
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BD Biosciences
CCAP
Eurofins MWG
Operon
Fisher Scientific UK
(NUNC & Nalgene)
Helena Biosciences
Invitrogen
Johnson & Johnson
Kemprotec Ltd
BD Biosciences, Edmund Halley Road, Oxford Science
Park, Oxford, OX4 4DQ, U.K.
CCAP, SAMS Research Services Ltd, Scottish Marine
Institute, Oban, Argyll, PA37 1QA, U.K.
Eurofins MWG Operon, Westway Estate, 28-32 Brunel
Road, Acton, London, W3 7XR, U.K.
Fisher Scientific UK, Bishop Meadow Road,
Loughborough, Leicestershire, LE11 5RJ, U.K.
Helena Biosciences Europe, Queensway South, Team
Valley Trading Estate, Gateshead, Tyne and Wear,
NE11 0SD, U.K.
Invitrogen Ltd, 3 Fountain Drive, Inchinnan Business
Park, Paisley, PA4 9RF, U.K.
Johnson & Johnson, 1 Johnson & Johnson Plaza, New
Brunswick, New Jersey, 08933, USA
Kemprotec Ltd, 11 Pennyman Green, Maltby,
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Lab MTM
LSHTM
Lund University
Millipore
Molekula
Moorfields Eye
Hospital
Oxoid Limited
Pall Life Sciences
Lab M, Topley House, 52 Wash Lane, Bury, BL9 6AS,
U.K.
London School of Hygiene and Tropical Medicine,
Keppel Street, London, WC1E 7HT, U.K.
Lund University, Division of Medical Microbiology, Hs
32, Medicinsk Mikrobiologi, Paradisgaten 2, Lund,
Sweden.
Millipore, Units 3 & 5, The Courtyards, Hatters Lane,
Watford, WD18 8YH, U.K.
Molekula Ltd, 6A Arrow Trading Estate, Corporation
Road, Audenshaw, Manchester, M34 5LR, U.K.
Moorfields Eye Hospital NHS Foundation Trust, 162
City Road, London, EC1V 2PD, U.K.
Oxoid Ltd, Wade Road, Basingstoke, Hampshire, RG24
8PW, U.K.
Pall Life Sciences, 5 Harbourgate Business Park,
Southampton Road, Portsmouth, PO6 4BQ, U.K.
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PNACL
Promega UK
Puritan Medical
Products
Qiagen Ltd
Sanyo Gallenkamp
Sanofi-Aventis
Sigma-Aldrich
Syngene
PNACL, The University of Leicester, University Road,
Leicester, LE1 7RH, U.K.
Promega UK, Delta House, Chilworth Science Park,
Southampton, SO16 7NS, U.K.
Puritan Medical products company LLC, Guilford,
Maine, USA
Qiagen Ltd, QIAGEN House, Flemming Way, Crawley,
W. Sussex, RH10 9NQ, U.K.
Sanyo E & E Europe BV, Biomedical Division, (U.K.
Office), 9 The office Village, North Road, Loughborough,
Leicestershire, LE11 1QT, U.K.
Sanofi-Aventis, 1 Onslow Street, Guildford, Surrey,
GU1 4XS, U.K.
Sigma-Aldrich Company Ltd, The Old Brick Yard, New
Road, Gillingham, Dorset, SP8 4XT, U.K.
Syngene, Beacon House, Nuffield Road, Cambridge,
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University of Leicester
VWR
The University of Leicester, University Road, Leicester,
LE1 7RH, U.K.
VWR International LTD, Hunter Boulevard, Magna
Park, Lutterworth, LE17 4NX, U.K.
Page 22
Introduction 1
1 INTRODUCTION
1.1 Free-living amoebae
Amoebae are both ecologically and medically important, and are named
from the Greek word for change (amoibe), referring to their continual changing
shape as they move and feed.
This ecologically important and diverse group are ubiquitous throughout the
environment. Within soil ecosystems naked amoebae consume large numbers of
bacteria, and are thought to have a key role (Rodriguez-Zaragoza, 1994) comparable
to that of flagellates within aquatic systems (Ekelund and Ronn, 1994). Several
amoebae species are significant within marine ecosystems as both consumers and
producers, and some are known to harbour symbiotic algae (Gast et al., 2009).
There are also free-living amoebae (FLA) that have the potential to live a parasitic
lifestyle and are called amphizoic in recognition of their endozoic existence (Page,
1988). Studies of these parasitic amoebae began in the late 19th
century with the
enteric pathogen Entamoeba histolytica (Lesh, 1975).
Amoebae are a group of unicellular eukaryotic microorganisms that include
Chaos carolinense the giant amoebae, the prototype Amoebe proteus, and parasites
such as E. histolytica, Naegleria fowleri and Acanthamoeba spp. Amoebae are
found throughout the environment from pole to pole with habitats including soil,
sand, seawater and freshwater.
Historically its taxonomy has been unsettled and variable, and this continues
today despite new data from a series of recent genomic sequencing studies (De
Jonckheere, 2004, Gast et al., 1996, Horn et al., 1999, Nassonova et al., 2010,
Schroeder-Diedrich et al., 1998, Smirnov et al., 2007, Stothard et al., 1998,
Weekers et al., 1994). Under the original taxonomic system, the phylum protozoa,
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Introduction 2
was divided into four groups with amoebae in sarcodina. However a second system
has been proposed classifying eukaryotes into six supergroups, with amoebae in
Amoebozoa (Adl et al., 2005). All taxonomic classification difficulties are further
compounded because amoebae are a polyphyletic group, having arisen from
different branches of the protozoal evolutionary tree (Schuster and Visvesvara,
2004).
Amoebozoa (Lühe, 1913, emend (Cavalier-Smith, 1998)) although
morphological diverse has several distinguishing features. Pseudopodia (Greek:
false feet) are extended from the cell surface to achieve the distinctive amoeboid
locomotion: they are non-eruptive and morphologically variable. Groups commonly
have subpseudopodia. They can be naked and surrounded by only a plasma
membrane, or testate with a partial single-chambered protective layer called a test.
They are usually uninucleate, rarely binucleate but occasionally multinucleate. Most
groups are at least dimorphic and able form protective cysts when environmental
conditions become adverse.
Pseudopodia are used for both locomotion and feeding: As a chemotaxis
response they detect concentration gradients of nutrients and other substances in
their surrounding environment. The pseudopodia extend and retract with
cytoplasmic streaming (Allen, 1961, Taylor, 1977) which results in the flow of
organelles through the cytoplasm within the cell. Only in the trophic form, can the
amoebae move and feed on surrounding nutrients and microorganisms by
phagocytosis. Pseudopodia surround the food and engulf it, before digestion occurs
within a phagolysosome. The cyst phase is a dormant resting stage, maintained until
the surrounding environment becomes favourable again.
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Introduction 3
Several amoebae spp. have been recognised as models for studying cell
mechanisms, one such species is A. castellanii, (ATCC 30010, Neff strain), which
has been used extensively. However a variety of different species have been used to
investigate and research areas including, means of locomotion (Allen et al., 1965,
Klopocka et al., 2009, Pollard, 1981, Sinard et al., 1989), phagocytosis (Obaray and
Coakley, 2001), chemotaxis response (Levchenko and Iglesias, 2002), intracellular
communication (Mogoa et al., 2010, Prusch and Roscoe, 1993) and cell
differentiation (Eichinger et al., 1999). The ability to encyst within a protective
outer layer when conditions become adverse provides protection from desiccation,
starvation, temperature extremes and many chemical disinfectants. Some groups
specifically Naegleria, can also have a flagellate stage.
In some circumstances, amoebae may act as environmental reservoirs, and
be exploited to take up microorganisms, which survive phagocytosis and multiply
within the amoebae (Axelsson-Olsson et al., 2005). Associations such as these may
represent how bacteria adapted and evolved to survive within eukaryotic cells
(Molmeret et al., 2005). Amoebae have even been described as the Trojan horse of
some human diseases (Alsam et al., 2006, Barker and Brown, 1994, Greub and
Raoult, 2004): With some of these microorganisms becoming more virulent whilst
within their amoebae host (Cirillo et al., 1997).
Acanthamoeba can act as a host to Staphylococcus aureus (MRSA) (Huws
et al., 2005), Legionella spp. (Rowbotham, 1998), Clamydia-related symbiont
(Horn and Wagner, 2004), Cryptococccus neoforans (Steenbergen et al., 2001), L.
monocytogenes (Ly and Muller, 1990), Mycobacterium spp. (Adekambi et al.,
2006), Compylobacter jejuni (Axelsson-Olsson et al., 2005), and Escherichia coli
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Introduction 4
serotype K-1 (Alsam et al., 2006) and serotype O157 (Barker et al., 1999), and may
act as a vector to transmit these pathogens to susceptible hosts, providing a route of
entry into the human body. The exact mechanisms of bacterial-amoebae interaction
are not understood. As amoebae feed on bacteria, how some pathogenic bacteria
survive within the amoebae, while other non-pathogens are killed, is unclear (Alsam
et al., 2006).
The term endosymbiotic is commonly used to describe microorganisms
within amoebae and their relationship to each other. However studies have shown
microorganisms do not represent true endosymbionts of amoeba, since they can be
both endosymbiotic or lytic, depending on environmental conditions, so the term
‘amoeba-resistant bacteria (ARB)’ has been coined as an alternative (Greub et al.,
2004).
Amoebae have increasingly been recognised as important pathogens, with
the cases of amebiasis increasing in frequency (Martinez, 1997). Infection occurs
largely in immunocompromised rather than otherwise healthy individuals.
Medically important amoebae that bridge the gap between parasitic and free-living
include Acanthamoeba spp., Balamuthia mandrillaris, N. fowleri, and most recently
Sappina pedata (Gelman et al., 2001, Qvarnstrom et al., 2009). Disease
pathophysiology helps to distinguish the infecting agent.
Acanthamoeba and Balamuthia are opportunistic pathogens, which can
cause devastating infections in immunocompromised individuals (either
granulomatous amoebic encephalitis (GAE) or cutaneous infections).
Acanthamoeba can also cause a non-opportunistic ocular infection known as
amoebic (or acanthamoeba) keratitis (AK). Naegleria fowleri causes a fatal non-
opportunistic meningoencephalitis known as primary amoebic meningoencephalitis
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Introduction 5
(PAM). Since the discovery of encephalitis caused by S. pedata in an otherwise
healthy young man is so recent, this suggests that there are probably other amoebae
capable of causing fatal infections in humans (Qvarnstrom et al., 2009).
1.2 Acanthamoeba
Acanthamoeba was first observed as a contaminant of trypsinised monkey-
kidney cells in tissue cultures in 1957 (Jahnes et al., 1957), but it was not until the
following year that the pathogenic potential of these amoebae was uncovered during
the development of a polio vaccine (Culbertson et al., 1958). Culbertson and
colleagues used contaminated monkey-kidney cell culture fluids to inoculate
immunosuppressed monkeys and mice, resulting in fatal encephalitis or
encephalomyelitis of the hosts within days. Initially the contaminant was thought to
be an unknown virus. However, upon histological examinations of the monkeys and
mice, and microscopy of the culture fluids the contaminant was identified as
Acanthamoeba. The first reported human infection by Acanthamoeba was described
in 1972 (Jager and Stamm, 1972). It was not until almost a decade later that
Acanthamoeba was characterised as an opportunistic organism, which infects
debilitated or chronically ill patients (Martinez, 1980).
These parasitic FLA are not as well adapted to parasitism as classic parasitic
protozoa such as Plasmodium spp. and Leishmania spp. Not only do the amoebae
invariably kill their host, but also they have not yet evolved to survive within them
for long enough to ensure transmission to a new host. Consequently host-to-host
transmission of these amoebic diseases has not yet occurred.
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Introduction 6
Acanthamoeba are incredibly versatile and resilient, as is demonstrated by
their ubiquitous distribution throughout nature and man-made habitats. Within soil
and water samples they are documented as being the most common amoebae if not
most common protozoan (Page, 1988), and they are found almost everywhere there
is water. Their tolerance of marked extremes in temperature, osmolarity, oxygen
availability, water potential and pH allows them to survive in such a wide variety of
locations. As does their ability to encyst when conditions become especially adverse
and survival is threatened.
Such universal distribution means they are not dependent on a host for
transmission and spread. Surprisingly, despite the potential opportunities, infection
of humans by Acanthamoeba is rare, and largely limited to immunocompromised
hosts. Acanthamoeba are responsible for several forms of disease. They can infect
eyes and cause Acanthamoeba Keratitis (AK) (often occurring in healthy
individuals), as well as cutaneous infections, and disseminated infections of the
lungs, the central nervous system (CNS), and the brain, known as Granulomatous
Amoebic Encephalitis (GAE).
So far today there are approximately 24 named species of Acanthamoeba,
but not all appear to be pathogenic (Visvesvara et al., 2007b). The first to be
isolated was A. polyphaga from dust, it was assigned the name Amoeba polyhagus
(Puschkarew, 1913), but was later re-described, and named A. polyphaga (Page,
1967). A. castellanii was discovered as a contaminant of a yeast culture of
Cryptococccus pararoseus (Castellani, 1930 ), which was later named
Hartmannella castellanii (Douglas, 1930). In 1931, the genus Acanthamoeba was
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Introduction 7
established (Volkonsky, 1931) and ultimately distinguished from Hartmannella.
Only then was H. castellanii reclassified as A. castellanii (Volkonsky, 1931).
The genus continued to be the subject of controversy, and was revised,
discarded, and finally redefined: But this time the earlier defining characteristics of
pointed spindles at mitosis, general form, locomotion and the appearance of cysts,
were replaced by more definitive features of cyst structure and presence of
acanthapodia (also known as fine finger-like pseudopodia) (Page, 1967).
As taxonomic techniques have become more refined there is now less
movement of species within the grouping system, which has stopped the historical
practice of using the genus names Acanthamoeba and Hartmannella
interchangeably. Hartmannella is a distinctly different amoeba (Page, 1967), which
has never been reliably associated with human pathologies (Cleland et al., 1982,
Culbertson, 1971, Jager and Stamm, 1972).
1.3 Acanthamoeba ecology
Acanthamoeba are dimorphic, and exist as either a cyst or as a trophozoite.
The trophozoites are the larger active feeding form, and ranges in size from 15 µm
to 45 µm. Distinguishing features of trophic amoebae are fine finger-like
pseudopodia, a nucleus containing a distinctive centrally located nucleolus, and a
contractile vacuole found in the cytoplasm integral to maintaining osmotic
equilibrium. The cytoplasm is granular and contains many mitochondria, lysosomes,
ribosomes, and contractile vacuoles. Cysts are smaller than trophozoites and range
from 10 µm to 25 µm. They are non-motile, resistant forms protected by an obvious
inner and outer wall, and are formed in response to a hostile environment. Amoebae
can exist as a cyst for many years if necessary until more favourable conditions
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Introduction 8
return. To continue trophic growth, dormant amoebae emerge through a pore or
ostiole, located on the cell wall at the junction of the ectocyst and endocyst
(Martinez, 1997), and covered by a protective flap or operculum.
Acanthamoeba are extremely abundant throughout nature and found
everywhere associated with water, lakes, ponds, oceans, in soil, and even dust
within the air. They have also been found in many man-made environments,
including baths, heating/ventilation/air conditioning systems, cold water storage
tanks, swimming pools, cooling towers of electric and nuclear power plants,
humidifiers, Jacuzzi tubs, dialysis machines, hospital hydrotherapy pools, dental
irrigation units, bottled water, contact lens solutions, bacterial, fungal and
mammalian cell cultures, and medical equipment (Martinez, 1997, Schuster and
Visvesvara, 2004, Visvesvara et al., 2007b). They have even been isolated from ear
discharge, skin lesions, corneal biopsies, cerebrospinal fluid (CSF), pulmonary
secretions, mandibular autografts, brain necropsies, and nasophayngeal mucosa.
Acanthamoeba can inhabit such a wide variety of locations because they are
tolerant of marked extremes in temperature, osmolarity, oxygen availability, water
potential and pH. Additionally they have the ability to encyst when conditions
become especially adverse and survival is threatened. Studies have shown cysts can
survive freeze thaw refreeze cryotherapeutic methods reaching temperatures of
between -50 to -130C (Meisler et al., 1986) although results from a later study
suggest that cyst survival is inversely linked to rate of freezing (Matoba et al.,
1989). Acanthamoeba cysts can also endure periods of up to 8 months at low
temperatures of –10, 4, 10 and 15C (Biddick et al., 1984), pH 2.0, moist heat of
Page 30
Introduction 9
60C for 60 min (Kilvington, 1991), gamma irradiation (250 K rads) and ultraviolet
radiation (800 mJ/cm2) (Aksozek et al., 2002).
Acanthamoeba have also been shown to remain viable over extended periods
of time. Cysts stored in a state of desiccation for 20 years have hatched and resumed
trophic growth (Sriram et al., 2008).
However some success has been shown in inactivating Acanthamoeba cysts
using solar disinfection (a simulated global solar irradiance of 850 WM-2
) providing
the water containing the organisms reaches a temperature of between 50-55C for 6
or 4 hours respectively (Heaselgrave et al., 2006).
Several environmental factors govern the distribution of Acanthamoeba,
namely organic matter. Bacteria must be readily available for the amoebae to feed
upon and thrive. Consequently Acanthamoeba flourish within biofilm, where food is
in continuous supply. Temperature is also significant, and must be optimal for
strains to thrive; however most can withstand a broad range. Some strains,
particularly clinical isolates, have enhanced growth at warmer temperatures
(>37C). Those isolated from corneal infections generally have an optimal growth
temperature of ~30C (Schuster and Visvesvara, 1998). Pathogenic strains of
Acanthamoeba able to survive within mammals must be able to actively grow at
temperatures of at least 37C. Thermotolerant strains have been isolated from many
biotopes. Including soil samples collected from the island of Tenerife, Canary
Islands, Spain, which showed 90.6% (39 of the 43 isolates) of those collected,
exhibited thermotolerance at 37C (Lorenzo-Morales et al., 2005). While samples
collected from soil in Talbot County, Maryland, USA showed 81.9% (17 of the 21
isolates) displayed thermotolerance between 37-39C (Sawyer, 1989).
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Introduction 10
Thermotolerance is not compulsory for species that infect the cornea, as
corneal temperature is around 5C lower than the body between 32-35C,
consequently most species of Acanthamoeba have the potential to become
opportunistic and colonise the corneal surface.
1.4 Acanthamoeba cultivation
As a general rule Acanthamoeba isolates adapt readily to axenic growth, and
as a consequence are an attractive model to use for morphological, biochemical,
nutritional and molecular studies: Often with the type strain A. castellanii Neff
(ATCC 30010) as the most popular choice of species.
Axenic growth for Acanthamoeba is established through a series of steps
based on the ‘walk out’ method (Neff, 1958). In this method amoebae are isolated in
vitro on non-nutrient agar (NNA) plates covered with a monolayer or streak of
gram-negative bacterial food such as E. coli or Enterobacter aerogenes. The
bacteria do not have a food source present and so cannot multiply. Any amoebae
present will crawl as they feed on the bacteria, producing plaque-like clearings in
the bacterial lawn during early growth stage. Once the food source/bacteria has been
exhausted the Acanthamoeba will encyst.
Providing the plates are sealed so they cannot dry out and are stored at 4C,
the cysts will remain viable for long periods of time. Additionally fresh amoebae
cultures can be maintained within the laboratory indefinitely, by transplanting a
small piece of agar containing trophozoites or cysts on to a fresh bacteria-seeded
NNA plate, and placing it isolate-side down. However it must be noted that this
method does not guarantee to establish monocultures of all isolates in a mixed
Page 32
Introduction 11
sample, as the slower growing amoebae are likely to be outrun by any faster
dividing amoebae.
To establish axenic growth, amoebae are dislodged from the plate surface
using a weak saline solution and are washed by centrifugation removing extraneous
bacteria, before being placed into sterile flasks containing liquid culture medium
and antibiotics. The most effective combination of antibiotics used to inhibit
residual bacteria within this enriched culture medium is penicillin and streptomycin,
or gentamycin. This method works for most isolates but some, in particular clinical
specimens, may require additional nutrients such as foetal calf serum and further
vitamins (Schuster, 2002).
There are several considerations that should be made when designing
experiments using a strain of Acanthamoeba that has been cultured for many
generations. As several studies have demonstrated that traits of strains can change
over long-term culture. It has been shown that strains can develop a reduced
tolerance to temperature, after long-term axenic culture (Pumidonming et al., 2010).
While some exhibit a reduced resistance to therapeutic agents, shown by the
comparison of two cultures of A. polyphaga Ros (one having been in continuous
culture since 1991, and the other cryopreserved since 1991), both tested against a
multipurpose contact lens solution (containing polyhexamethylene biguanide
(PHMB) 1mg/ml) (Hughes et al., 2003).
There may be many other traits that could also have been lost when cultured
for a substantial period of time in an artificial environment. In the case of assessing
the efficacies of contact lens disinfectant solutions against bacteria and fungi a limit
has been set, ensuring strains are passaged no more than five times from the original
Page 33
Introduction 12
culture, perhaps a similar limit should be taken into consideration when
experimenting with Acanthamoeba.
1.5 Acanthamoeba epidemiology
There are surprisingly few cases of Acanthamoeba infections considering the
ubiquitous nature of the amoebae and the opportunities for contact. However,
unsurprisingly it has been shown that more than 80% of the normal population
possesses anti-Acanthamoeba antibodies (Chappell et al., 2001, Cursons et al.,
1980): Allowing most typically healthy individuals the ability to combat
Acanthamoeba and not develop an infection when exposed to the amoebae. For an
infection to become established, specific predisposing factors must occur.
Breaks in the skin are the most likely mode of infection into the body.
However Acanthamoeba have been isolated from the nasal mucosa of healthy
individuals (Cerva et al., 1973), probably carried there as cysts on air currents or the
wind (Rodriguez-Zaragoza, 1994). So Acanthamoeba may be able to invade through
the upper respiratory tract, again given the right circumstances (Schuster and
Visvesvara, 2004). However the disease incubation period is unknown and weeks to
months may pass following an infection before the symptoms become apparent. As
a consequence of this time delay and the wide distribution of the amoebae within
the environment the precise portal of infection is masked. Once inside the body,
amoebae can spread through the blood to organs and the CNS.
Acanthamoeba infections within the body can result in a host of diseases
including GAE, nasopharyngeal, cutaneous, and disseminated infections, in addition
to AK affecting the eye. GAE is an opportunistic disease, affecting
immunocompromised individuals and therefore has no pattern of seasonality and
Page 34
Introduction 13
can occur at anytime of the year. However the incidence of GAE has increased with
the HIV/AIDS epidemic, because as a consequence more people are now
immunosuppressed and are therefore susceptible.
Acanthamebiasis primarily occurs in debilitated often chronically ill patients
who are immunocompromised or immunosuppressed often with HIV/AIDS, or who
have undergone organ transplants.
However some cases have been described in individuals who seem not to be
immunocompromised (Singhal et al., 2001). Acanthamebiasis has also been
reported in horses (Kinde et al., 2007), dogs (Dubey et al., 2005, Pearce et al.,
1985), a toucan (from which the etiological agent was isolated, cultured, grown at
44C and therefore shown to be thermotolerant, and genotyped to the T4 group
(Visvesvara et al., 2007a), turkeys, sheep, a kangaroo, reptiles, amphibians, fish
(Dykova and Lom, 2004), invertebrates, pigs, rabbits, pigeons and cattle (Cerva,
1981, Cirillo et al., 1997, Kadlec, 1978, Schuster and Visvesvara, 2004).
Models for encephalitis using the mouse as host have provided information
on virulence, epidemiology and the course of the disease (Mazur et al., 1995). Not
all species, strains and isolates of Acanthamoeba are pathogenic, and those that are,
vary in their virulence. Acanthamoeba virulence is measured by the period of time
from inoculation to the onset of symptoms leading to death, and the number of
animals that have died as a consequence, as well as the dose of inoculants required
to cause encephalitis (Schuster and Visvesvara, 2004). Virulence is also indicated
by the ability of Acanthamoeba to cause cytopathology in tissue cultures (Cursons
et al., 1980, De Jonckheere, 1980, Niszl et al., 1998).
Some strains maintained in axenic conditions for prolonged periods of time,
have been shown to lose their virulence (Mazur et al., 1995) (although this is not
Page 35
Introduction 14
always the case (Niszl et al., 1998)), their encystment capacity, and change their
susceptibility to drug treatments (Hughes et al., 2003). However most attenuated
characteristics including virulence can be restored if passaged in series through
human Hep-2 cell monolayer (Kohsler et al., 2009) or in vivo via intranasal
inoculation into mice and ultimately the brain (Xuan et al., 2009).
Acanthamoeba is the etiological agent for AK. The disease can occur in
otherwise healthy individuals who almost always are contact lens wearers.
Observations made studying rat and mouse models have shown mode of infection
usually occurs following a trauma to the eye and then wearing contact lenses (Ren
and Wu, 2010). With poor contact lens (and lens case) hygiene being a major
contributing factor to acquiring the disease. Infection does not automatically occur
in both eyes. However, there has been one case of fatal GAE with associated uveitis
and pharyngitis recorded in the literature, but never a case of GAE having
developed from AK (Jones et al., 1975, Visvesvara, 2010).
The precise incidence of AK is unknown, as the disease is not notifiable,
however it has undoubtedly been on the increase as more people wear contact lenses
today than previously. There was a recent outbreak of AK detected in the USA in
2006. Initially the cases were identified within the Chicago area by Illinois
Department of Public Health, and brought to the attention of the Centers for Disease
Control and Prevention (CDC). Who performed several surveys, one of which was a
retrospective survey of ophthalmology centres across the USA. Their results
showed an increase in the number of culture-confirmed cases during 2004-2006
compared with 1999-2003, in ten centres over nine states. Following a national
outbreak investigation, analysis indicated the odds of ever having used the
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Introduction 15
multipurpose contact lens solution, Complete MoisturePlus (AMOCMP)
manufactured by Advanced Medical Optics (now known as Abbotts Medical
Optics) were 20 times greater for AK patients than for controls. Following
communication with the food and drug administration (FDA, AMO voluntarily
recalled AMOCMP worldwide (Verani et al., 2009).
1.6 Granulomatous amoebic encephalitis (GAE)
Acanthamoeba are one of the causative agents of granulomatous amoebic
encephalitis also known as GAE. It is a rare and often fatal infection, found most
commonly in immune compromised or severely debilitated individuals. GAE was
first observed in 1972 (Jager and Stamm, 1972) with less than 200 cases caused by
Acanthamoeba documented within the literature (Schuster and Visvesvara, 2004).
This however is likely to be a false representation, as these infections are difficult to
diagnose and distinguish from bacterial and other microbial infections even in the
first world.
Headaches, slight fever, behavioural abnormalities, personality changes, stiff
neck, nausea, hemiparesis, seizures, cranial nerve palsies, and typical signs of
localised encephalopathy are all symptoms of the chronic progressive disease (da
Rocha-Azevedo et al., 2009, Marciano-Cabral, 2003, Schuster and Visvesvara,
2004, Walochnik et al., 2008). As the clinical signs are not specific, the disease is
often misdiagnosed. Differential diagnosis includes bacterial meningitis, viral
encephalitis, neurocyticerosis, and brain tumors (da Rocha-Azevedo et al., 2009,
Matson et al., 1988, Ofori-Kwakye et al., 1986).
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Introduction 16
Diagnosis if made, is almost always post-mortem following brain tissue
biopsies, and indirect immunofluorescence (IIF) staining of tissue sections (Bloch
and Schuster, 2005, Schuster et al., 2006b, Schuster and Visvesvara, 2004).
Medical image scanning to view the brain, such as computerised
tomography (CT) and magnetic resonance imaging (MRI) can be used to visualise
the lesions caused by Acanthamoeba, but these lesions are not specific enough to
the disease to base a diagnosis on (McKellar et al., 2006).
A diagnosis of GAE has been made pre-mortem, and was achieved as a
result of positive Acanthamoeba-specific PCR of several biopsy tissue and fluid
specimens, including cerebrospinal fluid (CSF), bronchoalveolar lavage specimens
(BAL), skin, lung, and brain tissue (from the main lesion): All of which had been
Acanthamoeba culture negative when tested (Walochnik et al., 2008).
PCR is a proven invaluable diagnostic tool, used to detect Acanthamoeba
infections. A reliable primer pair designed and developed from the complete DNA
gene sequence of the 18S ribosomal gene (18S rDNA: Rns), known as JDP1 and
JDP2, have the ability to detect all known Acanthamoeba subgroups successfully,
including those from the environment, AK and GAE patients (Stothard et al., 1998).
These primers within a PCR assay produce a specific amplimer of around 500 bp,
and from this sequence variation has allowed the development of a highly sensitive
T-group typing system. The system has even been used to determine an
epidemiological association between a keratitis-causing strain of Acanthamoeba, the
patient, their contact lens storage case and their domestic water supply (Ledee et al.,
1996). On going studies have identified T-groups 1-15, with the majority of GAE
and AK causing amoebae in the T4 subgroup (Gast, 2001, Hewett, 2003, Horn et
al., 1999, Schroeder et al., 2001, Stothard et al., 1998).
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Introduction 17
T-group typing is not the only molecular diagnostic technique for detecting
Acanthamoeba. Comparison of sequence variation of the mitochondrial 16S rRNA
genes (Ledee et al., 2003), Restriction fragment length polymorphisms (RFLP)
nuclear and mitochondrial rRNA (Kilvington et al., 1991, Kilvington et al., 2004),
and fluorescent probes to hybridise with Acanthamoeba DNA (Stothard et al.,
1999). Real time PCR assays have also been highly successful at determining
Acanthamoeba infections; they are rapid and particularly sensitive, with the ability
to be effective even with a low concentration of template DNA (less than 10 cells)
(Qvarnstrom et al., 2006, Riviere et al., 2006).
In addition to the molecular diagnostic techniques available to identify
Acanthamoeba infections, there are also many molecular methods. Although rare,
amoebae can be identified in wet preparations of CSF samples or those that have
been giemsa or H & E stained (Marciano-Cabral, 2003, Martinez, 1997, Seijo
Martinez et al., 2000, Sharma et al., 1993). Identification of amoebae in both
trophozoite and cyst stages can also be made from biopsied patient samples, which
have been formalin-fixed or paraffin-embedded (da Rocha-Azevedo et al., 2009),
using techniques including immunohistochemistry and fluorescent microscopy.
Rabbit generated, anti-Acanthamoeba antibodies are incubated with patient samples,
followed by a secondary incubation with anti-rabbit IgG associated with a
fluorescent marker such as FITC, and observed under fluorescent microscopy
(Culbertson and Harper, 1984). More recently species-specific monoclonal
antibodies for A. castellanii, A. polyphaga, A. lenticulata, and A. culbertsoni have
been developed (Guarner et al., 2007).
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Introduction 18
Typically GAE infections occur in the CNS tissue, but may involve the
lungs (Khan, 2006). The incubation period is unknown, and weeks to months may
elapse before the onset of the disease becomes apparent. This time delay obscures
the precise route of entry of the amoebae into the body, but the most likely portal is
the skin, olfactory neuroepithelium (Visvesvara et al., 2007b, Walochnik et al.,
2008) and in specific circumstances orally (Thamprasert et al., 1993). Once inside
the body, the amoebae most likely gain access into the CNS and lungs by
hematogenous dissemination, or by passing directly through the neuroepithelium
(Marciano-Cabral, 2003).
Acanthamoeba can also cause lesions in the skin, but these are more often
reported in association with HIV positive patients (da Rocha-Azevedo et al., 2009,
Torno et al., 2000). Cutaneous acanthamebiasis presents with multiple hard
erythematous nodules, papules, or ulcers, across the surface of the body (May et al.,
1992). If they occur simultaneously with CNS symptoms their presence is often
indicative of an infection by Acanthamoeba (da Rocha-Azevedo et al., 2009, Khan,
2006).
Pathogenesis of GAE is extremely complex and not yet fully understood,
and as a result, prognosis is extremely poor. As the majority of people who contact
GAE are often immune compromised, debilitated and/or chronically ill, these
predisposing factors are further compounded by the difficulty associated with
diagnosis. Treatment of Acanthamoeba infections is further hampered by the
amoebae ability to encyst when environmental conditions are detrimental (such as
in the presence of treatment drugs) and remain so until conditions become more
favourable.
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Introduction 19
However several patients have been diagnosed early enough and have
subsequently been successful treated (Lackner et al., 2010, Seijo Martinez et al.,
2000, Walia et al., 2007, Walochnik et al., 2008). Each of these patients has been
treated with a different antimicrobial, as often one drug treatment used successfully
in one patient is ineffective in another. Several factors are influential to the
outcome of the disease, these include: how early drug treatments are initiated; host
immune status; infective dose of the amoebae; antimicrobial sensitivity of the strain
and its virulence (Schuster and Visvesvara, 2004).
Drug treatments used clinically include amphotericin B, azithromycin,
fluconazole, 5-fluorocytosine (flucytosine), pentamidine isethionate, meropenem,
linezolid, moxifloxacin, miltefosine, amikacin, voriconazole and sulfadiazine
treated (Lackner et al., 2010, Schuster and Visvesvara, 2004, Seijo Martinez et al.,
2000, Walia et al., 2007, Walochnik et al., 2008).
1.7 Acanthamoeba keratitis (AK)
Acanthamoeba keratitis (AK) is the sight threatening, acute, progressive and
extremely painful ulceration of the cornea caused by species of Acanthamoeba. The
infection almost always occurs in immune competent individuals who are contact
lens wearers, or who have minor corneal abrasions.
AK was first recognised in 1973 in a rancher from South Texas, USA, who
had a history of trauma in one eye (Jones et al., 1975). Since then, year on year the
numbers of cases reported within the literature have continued to rise, however the
infection is not classified as a notifiable disease. Contact lenses are a risk factor
associated with AK, primarily in the users of soft contact lenses (Stehr-Green et al.,
1989). Incidence levels for the USA are estimated at an annual incidence of 1-2
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Introduction 20
cases per million contact lens wearer (Schaumberg et al., 1998). Mean while the
estimates for the U.K. are higher at 1 in 30,000 contact lens users (Seal, 2003). In
one decade between 1990 and 2000, 180 cases from the U.K. were detailed in the
literature (Radford et al., 1995, Radford et al., 1998, Radford et al., 2002) with most
patients referred for treatment to Moorfields Eye Hospital, London, U.K.. Several
factors are likely to be linked to the observed increase in AK cases, namely the
better understanding of the infection we have now, which has led to an increase in
the number of correct diagnoses made, and the years on year increase in number of
people wearing contact lens.
Typically AK occurs in one eye only, but bilateral AK has been described,
often developing as a complication of the initial infection (Lee and Gotay, 2010,
Wilhelmus et al., 2008). Symptoms of AK are not specific initially, and include
severe eye pain, eye redness, photophobia, blurred vision, and a sensation of
something in the eye, along with excessive tearing. Clinical symptoms develop to
include corneal inflammation leading to the formation of a ring-like stromal
infiltrate, corneal oedema, and erosion of the corneal epithelial cells.
AK suffers experience disproportionate eye pain, which thought to be linked
to radial keratoneuritis and the trophozoites found along the corneal nerves, leading
to thickening and distortion of these nerves (Yoo et al., 2004). Later stages of the
infection can result in epithelial denudation, stromal necrosis (da Rocha-Azevedo et
al., 2009), nerve oedema and retinal detachment, and if miss-diagnosed or a delay in
treatment occurs, the infection will almost certainly lead to blindness as the necrotic
region spreads inwards (Niederkorn et al., 1999).
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Introduction 21
A waxing and waning of the clinical course is usually a good diagnostic
indicator of AK. This trend is caused because Acanthamoeba are often partly
susceptible to non-protozoan treatments. An additional potential complication of
diagnosis and therefore management is a secondary infection due to bacteria.
Differential diagnosis of AK should be considered, as keratitis caused by Herpes
simplex has dendriform-appearing lesions on the cornea (Martinez 1997), while
Pseudomonas aeruginosa, has a similar type of infiltration (Clarke and Niederkorn,
2006).
AK can be diagnosed using the same techniques as for GAE, however cyst
and trophozoite stages can also be detected and identified in ocular samples, either
from biopsies or less invasive corneal scrapes, however considerable expertise is a
necessity. The samples are smeared onto glass slides and visualised with bright field
microscopy. Or wet mount preparations can be made using 10% KOH (Bharathi et
al., 2006). Wet mounts stained with Giemsa or with H & E can be used to readily
detect cysts, while trophozoites are harder to distinguish, as they resemble
inflammatory cells (Bharathi et al., 2006, da Rocha-Azevedo et al., 2009). Again, as
with GAE, IIF, monoclonal antibodies and fluorescent microscopy can be used to
detect Acanthamoeba in corneal samples, contact lenses and lens cases (Inoue et al.,
1999, Leher et al., 1999, Mietz and Font, 1997). There are several fluorescent
stains effective against Acanthamoeba including calcofluor white, fungiflora,
Acridine orange, Periodic acid-Schiff (PAS), and Gomori methamine silver (GMS)
(Hahn et al., 1998, Inoue et al., 1999, Vemuganti et al., 2000, Wilhelmus et al.,
2008). Confocal microscopy has also been shown to be useful in detecting AK
Page 43
Introduction 22
(Vaddavalli et al., 2011), but specialised equipment and experienced observers are
required.
Molecular techniques highlighted for diagnosing GAE infections as effective
with AK. Such techniques have made it possible to trace the source of AK
infections back to taps in the patient’s home (Booton et al., 2002, Kilvington et al.,
2004, Ledee et al., 1996).
Acanthamoeba are able to infect a cornea and establish an infection, when
specific circumstances arise. If there is corneal trauma in association with
contaminated water, or the wearing of contact lenses maintained with a poor
hygiene routine. AK infections principally occur in individuals, who wear soft
contact lenses, although infections have been documented in some wearing hard
contact lenses as well (Moore et al., 1987, Srinivasan et al., 1993). Acanthamoeba
cyst more readily attach to soft contact lenses which have a higher water content
than rigid hard lenses (Sharma et al., 1995).
Epidemic factors also linked to AK are swimming in contact lenses (Radford
et al., 2002), a failure to maintain proper lens care i.e. using unsterile contact lens
solutions, inadequate disinfection of the lenses, not soaking the lenses for the full
amount of their recommended period, storing the lenses in a dirty case and/or
washing the lenses in homemade saline solutions (Martinez and Visvesvara, 1997,
Radford et al., 2002).
As a result of wearing contact lenses (hard or soft), minor abrasion of the
corneal epithelium can occur, allowing the infection to take hold (Visvesvara et al.,
2007b). Once Acanthamoeba trophozoites have adhered to the surface of the corneal
epithelial cells, mediated by mannose-bonding protein (MBP) expressed on the
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Introduction 23
surface of the amoeba, damage is inflicted by phagocytosis of the host cells (Clarke
and Niederkorn, 2006). In vitro studies with Acanthamoeba and rat B103
neuroblastoma cells, viewed by scanning and transmission electron microscopy,
have shown phagocytosis. Following contact between the two cells types,
membrane blebbing of the nerve cells occurred, leading to either lysis of the nerve
cell, or ingestion of the nerve cell via amebastome-like (food cups) and channelling
the ingested cell into intracytoplasmic food vacuoles (Pettit et al., 1996).
To ensure a good prognosis, early diagnosis and appropriate treatment of
AK is vital. The first successful treatment of AK was reported in 1985, using an
antimicrobial agent belonging to the aromatic diamidine group of compounds,
known as propamidine isethionate and available as Brolene 0.1% w/v (Sanofi-
Aventis, Guilford, U.K.). This used in combination with Neosporin® (Johnson &
Johnson, New Brunswick, USA), and was Europe’s first-line medical treatment
until the mid 1990’s. When resistance to propamidine isethionate was detected in
some strains of Acanthamoeba and studies showed it to be only weakly cysticidal
(Perrine et al., 1995). The search has continued for other superior homologues and
alternative drug treatments such as the bis-biguanides, alexidine and chlorhexidine.
Alexidine has a cytopathic affect on Acanthamoeba, not unlike chlorhexidine, with
10 mg/mL being affective against trophozoites and 100 mg/mL against cysts. In
vivo experiments carried out using Chinese hamster corneas showed alexidine to be
less toxic to corneal epithelial cells than chlorhexidine at 100 mg/mL (Alizadeh et
al., 2009). More recently in vitro studies have shown Acanthamoeba to be
susceptible to PHMB (Hughes et all 2003), showing consistent cysticidal activity.
Page 45
Introduction 24
Now the recommended treatment regimes for AK are chlorhexidine with
propamidine, or its polymeric equivalent PHMB originally with propamidine but
now combined with hexamidine isethionate (Hexomedine, Sanofi-Aventis) (Seal,
2003). The biguanides are a class of cationic disinfectants, and the topical use of
them is recommended as the only effective therapy for resistant encysted forms of
Acanthamoeba in vitro, and likely in vivo (Dart et al 2009). If the treatment is
administered promptly enough a response to AK is expected within one week, with
total eradication of the active infection to occur within eight weeks. A stark contrast
to the four months of treatment needed if using Brolene and neomycin (Seal, 2003).
If the infection has not responded to topical biguanide, the next treatment to
attempt is the use of steroids, however this is controversial (Dart et al., 2009). As a
last resort in extreme cases that have shown no signs of improvement therapeutic
keratoplasty is then recommend (Dart et al 2009).
1.8 Taxonomy and Classification
So far today there are approximately 24 named species of Acanthamoeba
based on morphological characteristics (Schuster and Visvesvara, 2004, Visvesvara
et al., 2007b). Several of which do have pathogenic potential, and include A.
polyphaga, A. rhysodes, A. quina, A. griffini, A. lugdunensis, A. castellanii, A.
culbertsoni, A. healyi and A. hatchetti (Ledee et al., 1996, Schaumberg et al., 1998,
Schuster and Visvesvara, 2004, Yu et al., 2004).
The genus Acanthamoeba, is divided into three morphological categories (I,
II, and III) based on distinguishing features of the cyst, including diameter, and
shape of both endocyst and ectocyst walls (Pussard and Pons, 1977). Today the
most comprehensive dichotomous key for morphological taxonomy is based upon
Page 46
Introduction 25
the three group system, and is nearly 20 years old (Page, 1988). Acanthamoeba
cysts are double walled, with an inner layer or endocyst composed of cellulose, and
an outer made up of lipids and proteins known as the ectocyst (Blanton and
Villemez, 1978). Group I amoebae have large cysts with a diameter of 18 µm or
more, with stellate endocysts and smooth or wrinkled ectocysts (Table 1). Amoebae
in group-II are the most common. This group is also the largest and includes the
majority of the potentially pathogenic species (Table 1). Their cyst diameter is 18
µm or less, with polyhedric, globular, ovoid, or stellate endocysts and wavy
ectocysts. Group III amoebae have cysts of less than 19 µm with ovoid or globular
endocysts and wavy or smooth ectocysts (Table 1). The reliability of using cyst
morphology as a taxonomic characteristic came into question once it was observed
that ionic concentration of the growth media can alter cyst morphology (Sawyer,
1971).
Immunological, biochemical and physiological methods have been used in the
past to attempt to type and identify Acanthamoeba species. Techniques used include
western blotting and immunofluorescence, however they have been inconclusive at
distinguishing between Acanthamoeba species.
More recently a system has been proposed to classify Acanthamoeba based
on 18S rDNA (Rns) gene phylogeny (Gast et al., 1996, Schroeder-Diedrich et al.,
1998). 18S ribosomal RNA is the structural RNA for the small component of the
eukaryotic cytoplasmic ribosome: Which has a slow evolutionary rate making it an
ideal candidate to study and base reconstruction of ancestral divergences on.
Acanthamoeba are grouped based on sequence variation into evolutionary clades.
Page 47
Introduction 26
So far 15 clades have been recognised, each containing species or complexes of
closely related species: T1-T12 (Gast et al., 1996, Schroeder-Diedrich et al., 1998),
T13 (Horn et al., 1999), T14 (Gast, 2001), and T15 (Hewett, 2003). The group T4
contains the majority of species with pathogenic potential (Stothard et al., 1998).
Table 1, shows examples of morphologically designated Acanthamoeba, and how
they are grouped according to T-group genotyping.
Table 1. Morphologically designated Acanthamoeba species assigned to T-groups
based on phylogenetic analysis of 18s sequence variations.
T
group
Species Strain ID Reference Morphological
group
T1 A. castellanii ATCC 50494 (Dudley et al.,
2005)
II
T2 A. palestinensis ATCC 30870 (Gast et al.,
1996)
III
T3 A. griffinii ATCC 30731 (Gast et al.,
1996)
II
T4 A. rhysodes
A. polyphaga
A. castellanii
A. lugdenensis
ATCC 50368
ATCC 30971
ATCC 50374
L3a
(Gast et al.,
1996)
(Stothard et al.,
1998)
(Gast et al.,
1996)
(Kong, 2009)
II
II
II
II
Page 48
Introduction 27
T5 A. lenticulata ATCC 30841 (Stothard et al.,
1998)
III
T6 A. palestinensis ATCC 50708 (Stothard et al.,
1998)
III
T7 A. astonyxis ATCC 30137 (Stothard et al.,
1998)
I
T8 A. tubiashi ATCC 30867 (Stothard et al.,
1998)
III
T9 A. comandoni ATCC 30135 (Stothard et al.,
1998)
I
T10 A. culbertsoni ATCC 30171 (Stothard et al.,
1998)
III
T11 A. hatchetti
A. stevensoni
Sawyer:NMFS
RB:F1
(Stothard et al.,
1998)
(Kong, 2009)
III
II
T12 A. healyi CDC1283:V013 (Stothard et al.,
1998)
III
T13 Acanthamoeba
sp. *
- (Horn et al.,
1999)
-
T14 Acanthamoeba
sp. *
- (Horn et al.,
1999)
-
T15 A. jacobsi ATCC 30732 (Hewett, 2003) III
*: Without morphological designation; -: Not assigned.
Page 49
Introduction 28
Still despite the recent advances in identification and typing of
Acanthamoeba and all the information available in the literature there are still many
uncertainties, confusions and complications to unravel. Which is unsurprising,
considering the potential margin of error through misidentification of isolates,
possible mislabelling of culture tubes, and/or cross-contamination of cultures. To
highlight just one example, a group have analysed mitochondrial DNA restriction
fragment length polymorphisms (mt RFLP), as well as sequences of both the
nuclear 18S rDNA and mitochondrial 16S rDNA, of four morphological group II
Acanthamoeba. From their results they have proposed that A. divionensis, A.
paradivionensis and A. mauritaniensis all be regarded as synonyms for A. rhysodes
(Liu et al., 2005).
1.9 Molecular biology of Acanthamoeba
There has been a lack of interest in studying the molecular biology of
Acanthamoeba, but with the advent of recombinant DNA techniques, genomes of
the organisms have now become more assessable for studies. The recent outbreaks
of AK, and the realisation that Acanthamoeba can harbour pathogenic bacteria, and
therefore serve as a vector in human infections, have all boosted the current interest
in Acanthamoeba.
Acanthamoeba replicate by mitosis (Jantzen et al., 1990), with the nuclear
membrane disappearing during division (Ma et al., 1990). As yet there has been no
evidence of sexual reproduction occurring in Acanthamoeba (Yin and Henney Jnr,
1997). Evidence suggests that nuclear chromosomes are numerous and small with
some ranging from 200 Kb to larger than 2 Mb (Byers, 1986, Byers et al., 1990,
Rimm et al., 1988). Acanthamoeba trophozoites normally possess one nucleus,
Page 50
Introduction 29
which is approximately 16.6% of the size of the trophozoite (Khan, 2006). The
nucleus does not contain any membrane bound sub components, but does contain a
large centrally located nucleolus, which is also a prominent morphological
characteristic.
The genome of A. castellanii Neff has been estimated at 33 Mb, and made
up of approximately 60% GC (Byers, 1986). With a DNA content most commonly
estimated at 1.28 pg/amoeba (Adam et al., 1969). Nuclear DNA content varies
throughout culture growth, and decreases by approximately 50% (in unagitated
cells) during the transition from log to post-log phase, and is most likely linked with
preparations of the cell to encyst (Byers et al., 1969).
Mitochondrial DNA content has also been shown to vary throughout culture,
with measurements of log phase A. castellanii Neff containing an average of 0.15
pg/amoeba, and reducing to 0.01-0.02 pg/amoeba during encystment (Byers, 1986).
In recent years the mitochondrial genome of Acanthamoeba has been fully
sequenced, as part of collaboration to explore mitochondrial genome organisation
and evolution within protists. Acanthamoeba mitochondrial genome is a circular
molecule consisting of 41.6 Kb, with an AT content of 70.6%. Which contains the
genes that code for both large and small subunit rDNA, 16 tRNA, 8 open reading
frames (ORF) of undetermined function and 33 proteins. All of these are found in
the same transcriptional orientation and make up 93.2% of the total sequence
(Burger et al., 1995). A peculiar feature of the mitochondrial genome of A.
castellanii is the presence of a single continuous ORF (cox1/2) encoding subunits 1
and 2 of cytochrome oxidase (COX1 and COX2) (Burger et al., 1995).
Phylogenetic trees based on sequences of nuclear rDNA, place A. castellanii
on a branch with, or near (as an out group to) green algae and land plants. But as
Page 51
Introduction 30
small subunit SSU rDNA databases have expanded, Acanthamoeba has moved
away from the algal and plant clade, to a sister branch of animals and fungi
(Wainright et al., 1993) or outside of the multicellular lineages altogether (Cavalier-
Smith, 1993). However one group has discovered evidence of horizontal gene
transfer between algal chloroplasts and Acanthamoeba mitochondria (Lonergan and
Gray 1994). By sequencing from the mitochondria of A. castellanii, a 7,778 bp
region containing single-copy large subunit (LSU) and small (SSU) rRNA genes,
they identified three group I introns within the LSU rRNA gene (rnl). The introns
are placed within highly conserved regions and each possesses a freestanding open
reading frame (ORF). One of the introns was found to be identical to that of the
single group I intron in the chloroplast rnl of green algae Chlamydomonas
reinhardtii, and structurally homologous within the core region and the ORF they
encode. Suggesting intron movement has occurred between mitochondria and
chloroplasts, either intracellularly in a photosynthetic, remote common ancestor of
A. castellanii and C. reinhardtii or, more recently as a result of an intercellular
exchange of genetic material (Lonergan and Gray 1994).
With the continued search for new data, this theory has developed as a result
of the complete sequencing of the Acanthamoeba mitochondrial genome. The
overall size of A. castellanii’s mt DNA, its gene content and organisation most
closely resembles that of the chlorophycean algae Prototheca wickerhamii, than C.
reinhardtii. Comparison of the mitochondria from all three organisms shows A.
castellanii and P. wickerhamii to have almost identical respiratory and ribosomal
protein genes, while C. reinhardtii does not encode any ribosomal proteins and
lacks several standard respiratory genes. The authors argue that the results can be
interpreted in two possible scenarios, either C. reinhardtii does not share a common
Page 52
Introduction 31
ancestry with land plants and A. castellanii, or more likely, that they do all share a
common ancestor, and C. reinhardtii has diverged radically and lost many of its
former gene content (Burger et al., 1995).
Ribosomes and their associated sequences have been extensively studied
throughout biology as a means to better understand evolutionary origins. As a
consequence the ribosomal biology of Acanthamoeba is one of the better-
understood areas of its molecular biology.
One of the most studied and frequently used genes throughout eukaryotic
biology is a component of the small ribosomal subunit 40S, known as the small
subunit (SSU) 18S rRNA. SSU 18S is the structural RNA for the small component
of the cytoplasmic ribosomes, and is therefore integral to protein synthesis in all
living cells. Molecular analysis using 18S data to understand evolutionary
divergences has become extremely popular. Several factors make SSU 18S such a
prime target for molecular analysis, both its slow evolutionary rate and repetition
throughout the genome provide ample template for PCR. Additionally, the gene is
usually flanked with highly conserved regions, therefore making it relatively easy to
locate at the outset of the studies, and so readily accessible.
Amoebae rDNA coding sequences are arranged as is typical for eukaryotic
ribosomal gene repeat units; they contain one set of 5' 18S, 5.8S and 28S 3' genes.
Between these genes and neighbouring sets are spacer regions or internally
transcribed spacers (ITS). Acanthamoeba rRNA repeats unit is 12 Kb, containing
approximately 600 copies (Byers et al., 1990), while the coding sequence for the
18S rRNA gene is 2,303 bp long.
Page 53
Introduction 32
Acanthamoeba nuclear SSU 18S rRNA genes (18S rDNA; Rns) have been
fairly extensively studied and are at present receiving much attention, in the hope of
understanding why some strains appear pathogenic and others do not. Group I
introns within the rRNA sequence have been found in A. griffinii and A. lenticulata
(Gast et al., 1994, Schroeder-Diedrich et al., 1998), increasing the size of their
nuclear rRNA genes to approximately 2,800 bp, compared with 2,300 bp. Sequence
analysis of the Rns has allowed the development of a classification scheme,
allowing Acanthamoeba to be typed into one of a possible 15 Rns genotypes known
as T-groups (Booton et al., 2005, Gast et al., 1994, Hewett, 2003, Stothard et al.,
1998).
Many amino acid sequences have been studied and published, these include
complete and/or partial, mRNA, RNA and genomic sequences. The literature and
GenBank are dominated by 18S sequences from many Acanthamoeba sp., but other
sequences available include: actin I (Nellen and Gallwitz, 1982); myosin heavy
chains I (Brzeska et al., 1999); 26S (Lai and Henney, 1993); 5S (Zwick et al.,
1991); lactate dehydrogenase-like (Watkins and Gray, 2006); mannose-binding
protein (Garate et al., 2004); polyubiquitin (Hu and Henney, 1997) and profilin I
and II (Pollard and Rimm, 1991).
Although slow to start, the field of Acanthamoeba molecular biology has
moved forwards rapidly. Evidence has recently been presented to include a new
genotype of 18S, T16 (Corsaro and Venditti, 2010). While data also suggests
analysis of microsatellites found within the ITS located between 18S and 5,8S,
could be a potential candidate to further distinguish within the clades, especially T4
(Kohsler et al., 2006).
Page 54
Introduction 33
1.10 Aims
The aims of this thesis will be to study the molecular biology of
Acanthamoeba with the aim of developing a genotyping system to better resolve the
Acanthamoeba genus. An improved genotyping system could prove invaluable for
epidemiological fingerprinting and species resolution in the currently muddled
group. The new system will be put to the test in a case study of an outbreak of GAE
within a Swedish hospital. The following chapters will first introduce the subject
being investigated, and then outline the aims, methods, and any results found,
before a discussion is presented.
Page 55
DNA typing of Acanthamoeba sp. 34
2 DNA TYPING OF ACANTHAMOEBA SP.
2.1 Introduction
The classic morphological typing system divides the Acanthamoeba genus
into three groups based on morphological characteristics of the cysts including size
and shape (Pussard and Pons, 1977): Group I species, have relatively large cysts
with a diameter of at least 18 µm, with distinctly stellate endocysts and smooth or
slightly wrinkled ectocysts. Group II Acanthamoeba have a diameter of 18 µm or
less, with polyhedric, globular, ovoid, or stellate endocyst and wavy ectocyst. While
those classified to group III, have cyst diameters of less than 19 µm with ovoid or
globular endocysts, and wavy or smooth ectocysts.
The reliability of using cyst morphology as a taxonomic characteristic came
into question with the observation that variations in the ionic concentration of the
growth media can alter cyst morphology (Sawyer, 1971, Stratford and Griffiths,
1978). As a consequence the morphological classification scheme has been
challenged by the use of isoenzyme electrophoretic patterns to study intragenetic
relationships: Results have been mixed, with some good correlations between
isoenzyme patterns and morphological groups (Moura et al., 1992), and those that
contradicted their morphological group designations’ (Daggett et al., 1985, De
Jonckheere, 1983).
Isolation, cultivation and subsequent microscopy were once the main
method of Acanthamoeba identification. However due to the ambiguities of the
morphological system, these techniques have now been superseded: Although they
remain a vital component, as both a useful method of diagnosis to genus level, and
to cultivate sufficient numbers of cells for downstream assays. Attempts have been
made to develop molecular diagnostic techniques for studying Acanthamoeba and
Page 56
DNA typing of Acanthamoeba sp. 35
its associated diseases: These include techniques such as PCR amplification of
nuclear SSU 18S (Gast et al., 1996, Gunderson and Sogin, 1986), mitochondrial
cytochrome oxidase subunit 1 and 2 (cox1/2) gene (Kilvington et al., 2004),
mitochondrial 16S rRNA genes (Chung et al., 1998), as well as PCR-restriction
fragment length polymorphism (PCR-RFLP) analysis of nuclear and/or
mitochondrial rDNA (Yu et al., 1999) and fluorescent probes to hybridise with
Acanthamoeba DNA (Stothard et al., 1999). Ultimately molecular developments
have resulted in a phylogenetic typing system, which is both genus and subgenus-
specific, allowing Acanthamoeba to be classified based on 18S sequence genotype:
This system can assign isolates representing all three morphological groups to one
of a possible 15 T-groups (Booton et al., 2005, Gast et al., 1994, Hewett, 2003,
Stothard et al., 1998), based on sequence similarities of SSU 18S rDNA. The T-
group system has proven to be a sensitive tool, with the ability to detect very small
numbers of trophic amoebae in samples (Schroeder et al., 2001). Eukaryotes have
many copies of rRNA genes found repeatedly throughout the genome, providing an
excess of template available for PCR.
Although the 18S sequence typing system for Acanthamoeba is highly
sensitive and useful, it is not without its own specific complications. Such as
multiple alleles, where some isolates have been found to have three alleles of 18S
(Ledee et al., 1998). A second complication is the presence of Group 1 introns in
some species, such as A. griffinii (S-7; ATCC 30731) and A. lenticulata (PD2S;
ATCC 30841) (Gast, 2001, Gast et al., 1994), which distorts interstrain
relationships. The 18S T-group genotyping system is also not particularly robust
and clumps together both disease causing strains, into single clades with non-
pathogenic environmental strains (Booton et al., 2005). Of the 15 recognised
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DNA typing of Acanthamoeba sp. 36
groups, T4 is by far the largest with little resolution provided between the isolates.
The development of a second system has been attempted to better resolve T4 using
mt 16S rRNA sequences, but was unsuccessful (Ledee et al., 2003).
For a second system to be developed successfully, a gene suitable for
phylogenetic comparisons needs a high level of diversity. A key enzyme in aerobic
metabolism is mitochondrial cytochrome oxidase: Which is a component of the
respiratory chain, and involved with transfer of electrons from cytochrome c to
oxygen. Unusually within Acanthamoeba this gene named cox1/2, encodes for both
subunits of cytochrome oxidase and specified by a single continuous ORF (Burger
et al., 1995). Cytochrome oxidase genes have already been used for phylogenetic
typing systems in a variety of different organisms, including crayfish (Yue et al.,
2008), Echinococcus granulosus (Villalobos et al., 2007) and Trypanosoma cruzi
(Burgos et al., 2008).
The mitochondrial gene cox1/2 is a suitable contender as an alternative
target for sequence analysis typing, to be used as a target for rapid and sensitive
diagnosis of AK. Mitochondrial DNA has a large copy number in Acanthamoeba,
approximately 3,300 for a log phase amoebae of the Neff strain (Byers, 1986), and
analysis has shown it to have a large degree of nucleotide variation. Phylogenetic
analysis using cox1/2 has already been used to differentiate eight patient isolates, as
well as for six of them, matching their sequence homology with their respective tap
water isolates (Kilvington et al., 2004).
Recent studies have shown multiple housekeeping genes can be
concatenated to form a single super-gene alignment for building more robust
phylogenetic trees, which provide better discrimination power (Devulder et al.,
2005, Gadagkar et al., 2005, Kurtzman and Robnett, 2007). The use of the
Page 58
DNA typing of Acanthamoeba sp. 37
concatenated gene approach within Acanthamoeba, using the well documented SSU
18S rDNA gene from the T-group system, in combination with another
housekeeping gene, such as mt cox1/2 could yield more accurate trees with greater
powers of discrimination and ultimately provide the potential to phylogenetically
classify the subgenus and improve the diagnosis of AK.
2.1.1 Aims
To investigate the phylogeny and relatedness of multiple Acanthamoeba
species including both environmental and disease causing strains, by amplifying and
analysing two distinct genes, the housekeeping gene mt cytochrome oxidase subunit
1 and 2 (cox1/2) gene, and the well documented SSU 18S rDNA (Rns) gene,
making comparisons between their genotypes and morphological classifications.
Attempts will then be made to use the concatenated gene approach, combining the
18S gene, with the cox1/2 gene, to yield more accurate trees with greater powers of
discrimination. With the ultimate aim of providing a tool to improve the
phylogenetic techniques to classify the subgenus and improve the diagnosis of AK,
ideally differentiating between pathogenic and non-pathogenic strains.
Page 59
DNA typing of Acanthamoeba sp. 38
2.2 Materials and Methods
2.2.1 Chemicals
Chemicals were obtained from Sigma-Aldrich (Gillingham, U.K.) unless
otherwise stated. They were all sterilised by either autoclaving or by passage
through a 0.2 µm- Acrodisc® syringe filter (Pall Life Sciences, Portsmouth, U.K.)
prior to use.
2.2.2 Organisms
All strains of Acanthamoeba used in this study are held within the culture
collection of Dr Simon Kilvington, Department of Infection, Immunity and
Inflammation, University of Leicester, Leicester, U.K (Table 2). Unique AK clinical
isolates were obtained from patients receiving treatment at Moorfields Eye Hospital,
London, U.K. Corneal scrapes were collected and kindly donated by Mr John Dart
of Moorfields Eye Hospital, London, U.K: Corneal scrapes were placed, stored and
transported on agar slopes, ready for recovery and culturing techniques to be carried
out at the University of Leicester under the guidance of Dr Simon Kilvington.
Table 2. Microorganisms used for these studies.
Species CCAP/
ATCC
Strain name T
group
Morph.
group
Source
A. astronyxis a 30137 Ray and Hayes T7 I Soil, California,
USA
Page 60
DNA typing of Acanthamoeba sp. 39
A. castellanii a 1501/1a
50373
Neff/ OS4-7B T4 II Soil, California,
USA
A. comandoni a 1501/5 - - - Soil, France
A. culbertsoni a 30171 Lilly A-1 [AC-
001]
T10 III Primary monkey
kidney tissue
culture, India
A. griffinii a 1501/4 - T3 II Marine beach,
Connecticut,
USA
A. hatchetti a 30730 Bh2 T11 II Sediment,
Baltimore
Harbour,
Maryland, USA
A. healyi a - CDC1283
VO13
T12 III GAE, brain,
Barbados, BWI
A. lenticulata a 30841 PD2S T5 III Swimming pool,
France
A.
palestinensis a
30870
1547/1
AC-014 T2 III Soil, Israel
A.
palestinensis a
50708
1501/3c
OX-1/2802 T6 III Swimming pool,
France
A. polyphaga a 1501/3g - - - AK, USA
A.
palestinensis a
1547/1 - - - -
Page 61
DNA typing of Acanthamoeba sp. 40
A. polyphaga a 30873
1501/3d
Nagington T4 II AK, UK
A. polyphaga a - Ros T4 II AK, UK
Acanthamoeba
sp. a
- Environmental
1
- - Cold water
storage tank, UK
Acanthamoeba
sp. c
- A Keratitis
(AK) 1 to 19
- - AK, UK
Acanthamoeba
sp. b
- AK95/1153 - - AK, UK
A. tubiashi a 30876 - T8 I Freshwater,
Maryland, USA
a Dr S. Kilvington, University of Leicester;
b Prof. D. Warhurst, London School of
Hygiene and Tropical Medicine (LSHTM); c Mr J. Dart, Moorfields Eye Hospital; -:
no information available.
2.2.3 Monoxenic culture of Acanthamoeba
Acanthamoeba trophozoites were cultured by monoxenic growth on non-
nutrient agar plates seeded with a lawn of E. coli (NNA-E. coli) (Page, 1988). The
NNA media was comprised of 1.5% plain agar (Agar No. 1, Lab M™, Bury U.K.)
and 1 tablet of ¼ strength Ringer’s solution per 500 ml of deionised water. The agar
mix was autoclaved at 121C for 15 minutes, and allowed to cool to 50C before
being poured into plates, and left to dry overnight at 37C.
Once dried, the agar surface is seeded with 2-3 drops of a dense suspension
of bacteria E. coli strain JM101 (ATCC 33876) (see 2.2.3.1). The bacteria are
spread evenly over the surface with a sterile bacteria spreader, and the plates left to
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DNA typing of Acanthamoeba sp. 41
dry at room temperature. Seeded plates remain viable for two weeks, if stored at
4C.
For routine maintenance of Acanthamoeba, amoebae seeded plates were
cultured in air at 32C in sealed polythene bags. Cultures were refreshed weekly by
excising a one cm2 region of agar from the leading edge of the plaque, containing
numerous trophozoites, and placing it face down onto the centre of a fresh NNA-E.
coli plate for incubation as described above. To isolate and clone single cysts from a
mixed culture, individual cysts were transferred by micro capillary manipulation on
to a fresh NNA-E. coli, and again incubated as above.
2.2.3.1 Preparation of E. coli food source stock
To make the stock suspension, E. coli was streaked on to a Luria-Bertani
(LB) agar plate using a sterile disposable loop (Fisher Scientific UK). The agar
plates made comprises of 7.5 g plain agar (Lab M™), and 12.5 g of Difco LB
powder (BD Biosciences, Oxford, U.K.) per 500 ml of distilled water, and
autoclaved at 121C for 15 minutes, and allowed to cool to 50C before being
poured into plates, and left to dry overnight at 37C (plates are viable for 2 weeks
when stored at 4C). Once inoculated, the LB plates were incubated overnight at
37C allowing colonies to form.
Distinct single colonies were picked with a sterile disposable loop (Fisher
Scientific U.K.), and transferred to a 175 cm2 tissue culture flask (Nunc- Fisher
Scientific U.K.) with 100 ml of LB broth (comprising of 12.5 g of Difco LB powder
(BD Biosciences) in 500 ml of distilled water, and autoclaved at 121°C for 15
minutes), and propagated overnight at 37C in a shaking incubator. The suspension
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DNA typing of Acanthamoeba sp. 42
of E. coli was harvested under aseptic conditions and transferred to two 50 ml
polypropylene tubes. The bacteria were pelleted by centrifugation at 2,000 x g for
30 minutes, and the supernatant discarded. Next, all culture media was removed
preventing additional multiplication of the bacteria, by resuspending and thereby
washing the pellet in ¼ strength Ringer’s solution. This wash step was carried out a
total of three times, with the final pellet being resuspended in 10 ml of ¼ strength
Ringer’s solution.
The stock suspension of bacteria as a food source can be stored at 4C for up
to two weeks.
2.2.4 Axenic culture of Acanthamoeba
Where possible, strains were adapted to axenic growth in a semi-defined
media (Hughes and Kilvington, 2001), comprising of 20 g Biosate peptone (BD), 5
g Glucose, 0.3 g Potassium dihydrogen orthophosphate (KH2PO4), 10 mg Vitamin
B12, 15 mg L-Methionine per 900 ml of double distilled water, and if necessary pH
was adjusted to 6.5-6.6 with 1 M sodium hydroxide (NaOH). The culture media was
divided in volumes of 225 ml and autoclaved at 121˚C for 15 minutes. Prior to use,
penicillin/streptomycin solution (to a final concentration of 100 U/ml and 0.1
mg/ml, respectively) was added to all the media aliquots. These were then made up
to 250 ml with either sterile distilled water, or 10% heat inactivated foetal calf
serum (Invitrogen Ltd, Paisley, U.K.) depending on the strain of Acanthamoeba
being cultured. The complete media can be stored at 4˚C, and used within one
month.
Before axenic culture could be obtained superfluous bacteria surrounding
the Acanthamoeba was removed by treating cysts with 2% (v/v) hydrochloric acid
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DNA typing of Acanthamoeba sp. 43
(HCl) for 24 hours (Kilvington and White, 1994). All traces of HCl were then
removed by washing the cysts in sterile deionised water and pelleting with
centrifugation at 1,000 x g for 3 minutes, this wash step is repeated three times.
Washed cysts are inoculated into flat-sided Nunclon™ Surface tissue culture tubes
(Fisher Scientific UK) containing 3 ml of culture media, and incubated in air at
32C. Under these conditions, excystation should occur and any emergent
trophozoites can adapt and replicate in the media. Axenic strains were successfully
maintained in 25 cm2 (small) tissue culture flasks (Nunc- Fisher Scientific UK).
Cultures were maintained on a weekly basis by harvesting the amoebae
under axenic conditions and leaving a seeding stock replenished with fresh media,
this ensured cells did not become over confluent and were typically maintained at
mid log phase when metabolism is greatest. When required, cell density was
assessed microscopically using a modified Fuchs Rosenthal haemocytometer (SLS,
Nottingham, U.K.) and adjusted accordingly. When a higher density of cells was
required, a seeding stock was inoculated into an appropriately sized tissue culture
flask (Nunc- Fisher Scientific UK) under axenic conditions and incubated at as
before.
2.2.5 Cryopreservation of Acanthamoeba
As a standard procedure Acanthamoeba isolates were cryopreserved to
maintain integrity and avoid potential subculturing cross contamination.
Axenic trophozoites were grown to late log phase (approximately 1 x 106), and
then pelleted by centrifugation (500 x g, for 5 minutes). The pellet was resuspended in
(heat- inactivated) foetal bovine serum (FBS) (Gibco®, Invitrogen, Paisley, U.K.)
supplemented with 5% DMSO and 0.5 ml volumes were transferred to cryogenic vials
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DNA typing of Acanthamoeba sp. 44
(Nalgene- Fisher Scientific UK). The vials were immediately placed in a
cryopreservation unit- 5100 Cryo 1C freezing container “Mr. Frosty”, (Nalgene-
Fisher Scientific UK). The commercially available unit is designed specifically for
freezing organisms in cryogenic vials, and contains a special compartment to house
propan-2-ol ensuring it surrounds the vials. The cryopreservation unit is placed on the
bottom shelf of an -80C freezer (Sanyo Gallenkamp, Loughborough, U.K.) for a
minimum of four hours. Once in the freezer the container and propan-2-ol controls the
cooling rate of the cells to 1C /minute when they are in the presence of a
cryoprotective agent commonly dimethyl sulfoxide (DMSO), therefore preventing
intracellular ice crystal formation and cellular damage.
The vials containing strains were then catalogued and plunged into liquid
nitrogen at -196C, for long-term storage.
Recovery of a cryopreserved strain was achieved by rapidly thawing the
relevant vial in a 37C water bath. Then inoculating the cells into fresh culture media
warmed to 32C; although not essential, the media was usually supplemented with
10% FBS. To reduce any toxicity caused by the DMSO the media was replaced after
six hours. After 24 hours incubation, the recovered trophozoites should have adhered
to the wall of the flat-sided Nunclon™ Surface tissue culture tube (Fisher Scientific
UK); again the culture media was replaced with a fresh supply. Confluent trophozoite
growth was usually observed after a further 48 hours incubation 32C.
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DNA typing of Acanthamoeba sp. 45
2.2.5.1 Cryopreservation of bacteria
Bacteria were cryopreserved, by suspending the cells in LB broth containing
5% DMSO and rapidly freezing catalogued vials directly at -80C (Sanyo) for long-
term storage. Cells were recovered by rapid thawing at 37C, and inoculated on to
appropriate agar media or broth for incubation at 37C. Again media was replaced
after six hours to reduce any toxicity caused by the DMSO.
2.2.6 Acanthamoeba DNA isolation
Axenic Acanthamoeba cultures were harvested for high quality genomic
DNA extraction by the UNSET method (Gast et al., 1994). If attempts to axenise
strains had failed, the cells were continued to be maintained and propagated by the
slower method of monoxenic culturing (section 2.2.3), Acanthamoeba cells and
subsequently DNA was collected directly from the agar surface, with resulting
lower concentrations of recovered DNA (see section 2.2.6.1).
Cells were harvested by centrifugation at 1,000 x g for 5 minutes, and the
supernatant gently decanted. Cells were resuspended in 10 ml of ice cold
Dulbecco’s phosphate buffered saline (dPBS) (one tablet (Oxoid, Basingstoke,
U.K.) dissolved in 100 ml, autoclaved at 121˚C for 15 minutes), and transferred to a
14 ml polypropylene centrifuge tube. Once washed, the cells were pelleted by
centrifugation at 1,000 x g for 5 minutes, and the supernatant removed. 3 ml of
UNSET buffer lysis was added (8M urea, 0.15M NaCl, 2% sarkosyl, 1mM EDTA,
0.1M Tris, per 1,000 ml of nH2O, adjusted to pH 8.0 if necessary) and the solution
gently inverted for approximately 10 seconds until lysate clears. Immediately DNA
was extracted with the addition of 3 ml of phenol-chloroform (1:1), and gently
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DNA typing of Acanthamoeba sp. 46
rocked from side to side ensuring the phases are completely mixed. The phases were
separated by centrifugation at 2,000 x g for 2 minutes and the lower phase removed,
using a fine tip pipette. These steps were repeated with a further 3 ml of phenol-
chloroform (1:1), and followed by 3 ml of chloroform: isoamyl alcohol (24:1). With
these steps culminating in a final spin of 2,000 x g for 15 minutes. The upper
aqueous phase then transferred to a fresh polypropylene tube, and 0.8 volumes of
ice-cold isopropanol added. The solution was gently mixed before being held at
room temperature for 30 minutes or overnight at 4˚C. All precipitated DNA was
pelleted by centrifugation at 2,000 x g for 15 minutes. The resulting pellet was
washed with 1 ml of 70% ethanol, the pellet dislodged and recentrifuged at 2,000 x g
for 5 minutes. All supernatant removed with a fine bore pipette. This ethanol wash
step was repeated twice more. After the final removal of the supernatant, all
remaining alcohol was evaporated in an air incubator at 32˚C (approximately 20
minutes) with the cap loosened. Ultimately the pellet was left to dissolve at 4˚C in
200-400 mL of TE0.1 buffer (10mM Tris HCl, 0.1mM EDTA, autoclaved at 121˚C,
and stored at 4˚C), containing RNase A (5 μl/ml) to remove any RNA.
The presence of the DNA was determined by running an aliquot on an
agarose TAE gel (see section 2.2.7): With DNA quantity obtained using GeneSnap
(version 7.08) (Syngene, Cambridge, U.K.) with the G: BOX XT gel documentation
system (Syngene).
2.2.6.1 DNA extraction from Acanthamoeba in monoxenic culture
To harvest Acanthamoeba from the surface of the NNA plates, cells must be
washed off the agar with 5 ml of ice cold dPBS, squirted repeatedly across the
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DNA typing of Acanthamoeba sp. 47
surface until the amoebae cells are dislodged. The solution is then transferred to a
14 ml polypropylene tube, and centrifuged at 1,000 x g for five minutes. The
supernatant was gently decanted, and the remaining amoebae washed within the
tube with 10 ml of ice-cold dPBS and centrifuged again. This wash stage was
repeated three times, to remove any remaining E. coli, before DNA extraction could
commence (section 2.2.6).
2.2.7 Agarose gel electrophoresis
PCR product/DNA samples were separated in 1.2% 1X-Tris-acetate (TAE)-
agarose gels (TAE 10X stock solution: 48.4 g Trizma base, 11.4 ml glacial acetic
acid, 20 ml EDTA (0.5 M, pH 8.0) per 1,000 ml nH2O) at 2 v/cm (approximately
one hour) containing ethidium bromide (EtBr) 0.5 μg/ml, and visualised with a G:
BOX XT gel documentation system (Syngene).
If required, a specific band can be isolated from a reaction containing multiple
bands. Specific sized DNA fragments can be separated directly from a lower
percentage agarose gel. Certain parameters must be followed to prevent degradation
and cutting of the DNA caused by prolonged exposure to UV light. The DNA
sample is separated in a 0.8% 1X-TAE agarose gel, at 2 v/cm (approximately one
hour) containing EtBr (0.5 μg/ml). Prior to UV exposure the gel is placed on a glass
plate, and the DNA fragment of interest is excised quickly with a sterile scalpel
blade, and transferred into a 1.5 ml eppendorf tube.
The quantity of DNA was obtained using GeneSnap (version 7.08) (Syngene)
with the G: BOX XT gel documentation system (Syngene).
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DNA typing of Acanthamoeba sp. 48
2.2.8 Primer design
To obtain gene sequences from such a broad range of Acanthamoeba
isolates several primer pairs were used. The JDP and 18s primers were obtained
directly from the literature, although modification was made to 18sR (SSU1) to
improve its specificity (Table 3), and the cytochrome oxidase (cox) primers were
designed specifically for this study (Table 3). Primers were identified using
GeneFisher software (Giegerich, 1996), and constructed by Eurofins MWG Operon
(Eurofins MWG Operon, London, U.K.). Following primer optimisation reactions,
basic PCR was carried out with the relevant primer specific parameters shown in
(Table 3).
2.2.9 PCR amplification of DNA
PCR amplification of DNA was performed in either a 96-well GeneAmp®
PCR System 9700 (Applied Biosystems, Warrington, U.K.) or a 24-well Perkin-
Elmer GeneAmp® PCR system 2400 (Applied Biosystems). Using either a standard
pre-made PCR reaction mixture (2X Reddymix™ PCR master mix; 1.5 mM MgCl2;
ABGene, Surrey, U.K.), or one prepared using individual components (ABGene).
All oligonucleotide primers were purchased from Eurofins MWG Operon.
To complete the pre-prepared PCR mix (2X Reddymix™; ABGene),
approximately 100 ng of genomic DNA template was added, as well as 1 μM of
both forward and reverse primers, and if necessary the MgCl2 concentration was
upwards adjusted from the initial concentration of 1.5 μM. Finally the mixture was
made up to a total volume of 50 μl PCR mix with nH2O.
The PCR mixture prepared with individual components consisted of
approximately 100 ng of genomic DNA with 1X reaction buffer, magnesium
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DNA typing of Acanthamoeba sp. 49
chloride (MgCl2) (1.5 mM - 2.5 mM), 400 μM of dNTP, 1 μM of each forward and
reverse primer, 1X sucrose dye (10X: 20% sucrose, 2 mM cresol red), and 1.5 U/μl
of Red Hot DNA Polymerase (ABGene, Surrey, U.K.). The mixture was then
increased to a total volume of 50 μl with nH2O.
Thermal cycling conditions varied according to the oligonucleotides used
within the reaction, but a typical cycle comprised of an initial DNA denaturing stage
at 95˚C for 5 minutes, followed by 35 cycles of 95˚C for 30 seconds, 50˚C - 62˚C
for 30 seconds (primer annealing depending on primer Tm), followed by 72˚C for
amplification of product. The extension time was dependent on the size of the
product being produced (30 seconds per 500 bp (Sambrook et al., 1989)). Followed
by a final extension step for 10 minutes at 72˚C, however if the product was to be
cloned in a TA vector this duration of this step was increased to 30 minutes. Finally
the reaction was held at 14˚C, before a small quantity of the product was analysed
by agarose gel electrophoresis (section 2.2.7).
If PCR products of the expected size were obtained the appropriate bands
were purified by one of the techniques in section 2.2.10.
2.2.10 Purification of PCR products
As a general rule, the PEG method (2.2.10.1) was used for screening
to confirm correct band size. While for purifying DNA ready for insertion into
plasmids, the use of Microcon® centrifugal filters (device YM-50) (Millipore) were
favoured as they produced the best sequencing results and therefore the most pure
DNA of the three methods (2.2.10.1.i). If multiple DNA bands were produced by
PCR method 2.2.10.2 was used to isolate the DNA of the correct size.
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DNA typing of Acanthamoeba sp. 50
2.2.10.1 PEG purification of single bands
Production of a single band allows target DNA to be precipitated directly
with a non-ionic water-soluble uncharged polymer such as polyethylene glycol
(PEG) (Cole, 1991), which can then be dissolved in nH2O. Or centrifuged in a
single-spin Microcon® centrifugal filter device (YM-50) (Millipore, Watford,
U.K.) that also results in sequencing or cloning grade DNA.
For the precipitation of DNA using PEG, 50 µl of 20% PEG (2.5 M NaCl)
(10 g PEG 8,000, 7.3 g NaCl, up to 50 ml with nH2O) was added to a 1.5 ml
eppendorf, with 45 µl of PCR mix, and mixed by repeated pipetting, before being
incubated for 15 minutes at 37C. The mixture was then centrifuged at 12,000 x g
for 15 minutes, and the supernatant carefully removed. The DNA pellet was washed
with 0.5 ml of cold (4C) 70% ethanol (EtOH), and again centrifuged at 12,000 x g
for 5 minutes. The EtOH was again carefully removed, with any remaining alcohol
allowed to evaporate during incubation at 32C. Once completely dried the purified
PCR products were dissolved in 20 µl of nH2O (by pipetting several times and
warming at 37C the process can be assisted). Recovery can be confirmed by
running 2 µl on an agarose gel (section 2.2.7).
2.2.10.1.i Micron®–PCR purification of single bands
The alternative method using Microcon®–PCR filter purification units
(Millipore), and the supplied instructions, results an average 95% recovery rate. The
system relies on an ultracel YM membrane and centrifugal force. The Microcon
sample reservoir was inserted into the vial, and the PCR solution carefully pipetted
into the reservoir (avoiding contact with the membrane), and the lid is sealed. The
assemblage was then centrifuged at 14,000 x g for 12 minutes, at 25C. Finally the
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DNA typing of Acanthamoeba sp. 51
sample reservoir was removed from the original vial, and transferred inverted, to a
new 1.5 ml eppendorf, and again centrifuged, but this time at 1,000 x g for 3
minutes, collecting the now purified PCR product into the base of the new
eppendorf.
2.2.10.2 Target DNA purification from a multiple band PCR
If PCR amplification resulted in multiple bands, purification by an
alternative method is necessary. Products from the reaction mix were separated in a
0.8% agarose gel, and the target band excised with a sterile scalpel. The gel segment
containing the band was heated to 60C with 3X 7 M guanidine acetate (67 g
Guanidine hydrochloride, 20 ml Potassium acetate 3 M, pH 4.8 (29.4 g Potassium
acetate, 11.5 ml Glacial acetic acid, to 100 ml with nH2O, autoclaved at 121C for
15 minutes, and stored at 4C), total volume was increased to 90 ml and the pH
adjusted to 5.5 with NaOH (10 M) if necessary), whilst warming and stirring.
Volume is then increased to 100 ml and the solution passed through a 0.45 µM filter
membrane), until the agarose gel dissolves. The chaotropic solute guanidine acetate
increases the entropy of the system and disrupts the hydrogen bonds of DNA,
forming a hydrophobic environment. Driven by this dehydration, when salt
concentrations are high, the positively charged salt ions form a salt bridge between
the negatively charged nucleic acids and the negatively charged silica. Proteins
metabolites and other contaminants do not bind to the silica with the nucleic acids
(Boom et al., 1990, Nelson, 1992, Vogelstein and Gillespie, 1979). Accordingly 20
µl of silica in nH2O (300 mg/ml) was added to the reaction (2 g silica suspended in
20 ml nH2O, and allowed to settle for two hours. The milky supernatant removed
again and the silica resuspended in a further 20 ml of nH2O, these steps repeated
twice more, before the remaining volume of silica was estimated, and the silica re-
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DNA typing of Acanthamoeba sp. 52
suspended in 2 volumes of nH2O). Ensuring it was fully in-suspension before doing
so. The PCR reaction, guanidine acetate and silica mixture was vortexed and
incubated at ambient temperature for 5 – 10 minutes with occasional further gentle
mixing. The mixture was centrifuged at 10,000 x g for 10 seconds and the
supernatant removed. The DNA bound silica was washed three times in 80%
isopropanol, spinning at 10,000 x g for 10 seconds and removing all supernatant,
between washes. Ensuring the eppendorf cap was opened, the DNA bound silica
was incubated at 37C for approximately 10 minutes ensuring the pellet was
completely air-dried. The DNA was then dissolved in 20 µl of pre-warmed nH2O,
and incubated for 5 minutes at 60C (with occasional mixing). Finally the silica was
removed by centrifuging at 10,000 x g for 1 minute, and the supernatant containing
the DNA was recovered in to a fresh eppendorf.
2.2.11 Ligation of DNA into cloning vectors
Ligation reactions were carried out in a 0.5 µl eppendorf. In a standard
reaction containing 3.5 µl of amplified cox1/2 or 18s PCR product, 5µl ligation
buffer (2X), 25 ng linearised pGEM®-T Easy vector (Promega UK, Southampton,
U.K.), 3 Weiss units of T4 DNA ligase (Promega UK), and nH2O to a final volume
of 10 µl were gently mixed by pipetting (so as not to form air bubbles). Ligation
mixtures were incubated at ambient temperature for one hour, or 48 hours at 14C.
A small aliquot of the modified plasmids were visualised and quantified by
1.5% agarose gel electrophoresis (detailed in section 2.2.7).
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DNA typing of Acanthamoeba sp. 53
2.2.12 Production of ultra competent E. coli cells
Ultra competent E. coli cells were produced for transformation using a
simplified method, based on the technique developed by Inoue (Inoue, 1990), and
produces cells with an efficiency of >108 cfu/µg of plasmid DNA.
One ml of an overnight culture of E. coli DH5α (ATCC 53868) was added to
a 2 L flask containing 250 ml of super optimal broth (SOB) (20 g Bacto™ tryptone
(BD Biosciences), 5 g Bacto™ Yeast extract (BD Biosciences), 0.5 g NaCl, per 950
ml of nH2O; Dissolved by shaking, then added 2.5 ml KCl (1M), adjusted to pH 7
with NaOH (5 M), and made up to 990 ml with nH2O: Autoclaved at 121C for 15
minutes, and finished by added 5 ml MgCL2 (2 M), and MgSO4 (2 M)).
Over a period of approximately two days, the cells were incubated at 18C in a
shake incubator (200-250 rpm), until an OD600 = 0.6 (optimal but 0.4 to 1 will
work). Once the correct OD had been reached, the cells were placed on ice for 10
minutes, before being pelleted at 2,500 x g for 10 minutes at 4C.
Next the cells were carefully resuspended without air bubbles in 80 ml of ice
cold transformation buffer (TB) (3.36 g PIPES, 2.2 g CaCl2, 18.64 g KCl, per 900
ml nH2O: pH adjusted to 6.7 with KOH (5 M), whilst slowly stirring 10.88 g
manganese chloride (MnCl2) was added: Volume adjusted to 1 L with nH2O: Filter
sterilised and stored at 4C). The cells were stored on ice for 10 minutes, before
being pelleted again at 2,500 x g for 10 minutes at 4C. Cells were carefully
resuspended (air bubble free) in 20 ml ice cold TB: 7% DMSO was added and the
solution placed on ice for 10 minutes. Aliquots of 0.5-1 ml quantities were
transferred to cooled cryotubes and frozen in liquid N2. The cells can be stored for
up to a one-year at –70C.
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DNA typing of Acanthamoeba sp. 54
2.2.13 Heat shock transformation of ultra competent E. coli
The fragile ultra competent E. coli were thawed slowly on ice, for
approximately 30 minutes. The solution was distributed in 200 µl aliquots, into
cooled 1.5 ml eppendorfs and placed on ice. Added to one aliquot was 100 ng of
circular pGEM®-T Easy plasmids now complete with inserts, while the control
aliquot had an addition of 2 µl of TE0.1 as a substitute for the lack of plasmid. The
tubes were incubated on ice for 30 minutes, with the occasional gentle mix. The
incubated solutions were exposed to a heat pulse without agitation at 42C for 45
seconds, and transferred to an ice bath for 2 minutes. Next is the addition of 0.8 ml
of SOC (SOB, with 20 mM filter sterilised 1 M glucose (super optimal broth with
catabolite repression)), and the cells are left to recover and multiply in a shaking
incubator (200-250 rpm) at 37C.
Following incubation, identification of successful transformants is carried
out on specific selective LB agar plates. A 200 µl aliquot of the transformation mix
and control were plated on to LB agar indicator plates containing ampicillin (100
μg/ml), which have been surface supplemented with the blue/white screening
indicator solutions, X-gal- 5-bromo-4-chloro-3-indolyl-β-D-galactoside in
dimethylformamide (80µg/ml), and Isopropylthio-β-D-galactoside in nH2O (IPTG)
(0.5mM): These solutions must be added respectively, with each being left for a
period of time to allow the liquid to evaporate. Once the surface is dry, the plates
can be inoculated with transformed bacteria.
When antibiotic pressure is required, the agar is heated until molten but
allowed to cool to 50C before the addition of the required quantity of antibiotic.
The bottle is then gently shaken to ensure even distribution throughout the solution.
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DNA typing of Acanthamoeba sp. 55
In addition, the remaining solution from the test transformation mix was
centrifuged, and the supernatant removed. The resultant pellet of cells was also
plated on to an LB agar ampicillin indicator plate. All seeded plates were incubated
overnight at 37C.
Multiple second round selections on selective indicator LB agar were made
of successful transformants to ensure single colonies are isolated. From this second
plate distinct colonies were selected for cultivation in LB broth (section 2.2.3.1)
with continued selective pressure of ampicillin at 100µg/ml, in a shake culture of
37C, revolving at 180 rpm.
2.2.14 Restriction enzyme digestion
In a 0.5 ml eppendorf, the following components were mixed to produce a
2X restriction enzyme digestion, 2 µl of nH2O, 1 µl of restriction enzyme EcoR 1
(Promega UK) and 2 µl of 10X enzyme buffer H (1X: 90mM Tris-HCl, 10mM
MgCl2, 50mM NaCl, pH 7.5) (Promega UK) (8-12 U/µl), (the enzyme must be kept
on ice until returned to -20C). Added to the reaction mixture was 15 µl of ligated
target DNA (pGEM®-T Easy plasmids with insert). The mixture was incubated for
4-12 hours, at 37C, and a small amount of the reaction was resolved by agarose gel
electrophoresis (as in section 2.2.7).
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DNA typing of Acanthamoeba sp. 56
Table 3. Primers (5' to 3') used for gene sequence typing of Acanthamoeba spp. SSU 18S rDNA and cox1/2, with corresponding PCR
parameters.
Primers Sequence MgCl2
conc. MM
Annealing
temp. C
Extension
time sec.
Product
size bp
Paper cited
Acanthamoeba 18S:
JDP1F
JDP2R
GGCCCAGATCGTTTACCGTGAA
TCTCACAAGCTGCTAGGGGAGTCA
1.5 60 45 450 (Schroeder et
al., 2001)
AC-892c GTCAGAGGTGAAATTCTTGG Sequencing primer
18sF
18sR
CTGGTTGATCCTGCCAG
Originally called SSU1
GATCCTTCTGCAGGTTCACCTAC
Modified from SSU1
1.5 48 90 1.5-2 kb (Weekers et
al., 1994)
Modified for
this study
18s588 CGCGCAAATTACCCAATC Sequencing primer
Acanthamoeba cox1/2:
CoxA125 ATGATTGGHGCTCCNGAYATGG 2.5 60 45 748 Designed for
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DNA typing of Acanthamoeba sp. 57
CoxA873 TGRCCTCCCCATAATGTAGC this study *
Cox1/2F
Cox1/2R
GAATTAGCTGCTCCGGGTTC
TCAGGATAATCGGGGATCCTTC
2 – 2.5 46 or 52 90 1.2 kb (Kilvington et
al., 2004)
CoxA-486F
CoxA-
1057R
GCHGGTGCTATTACTATGCTTT
CCWGCAAARAARGCAAAAACDGC
1.5 56 (60
touchdown)
30 566 Designed for
this study *
SeqCoxA GGTGCTATTACTATGC Sequencing primer
*Wobble nucleotides for degenerate primers were those according to Operon: H, A/C/T; W, A/T; R, A/G; N, A/T/G/C; Y, C/T.
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DNA typing of Acanthamoeba sp. 58
2.2.15 Plasmid purification
Several distinct white colonies were chosen at random from the LB agar
(ampicillin 100 μg/ml) indicator plates, seeded with pGEM®-T Easy (plus insert)
plasmids and transformed E. coli. A small section of each colony was inoculated
into 5 ml universal tubes containing LB broth (ampicillin 100 μg/ml), and
propagated overnight in a shaking incubator at 37C, revolving at 180 rpm.
Plasmids were then purified with the relevant method depending on their
downstream application.
For quick screening to confirm insert presence and size, the silica method
(section 2.2.15.i) was selected. If plasmids were required for sequencing, either the
comprehensive method (section 2.2.15.ii), or the plasmid purification kit (Qiagen
Ltd, Crawley, U.K.) (2.2.15.iii) was carried out. As a general rule the
comprehensive method was trialled first, but if failed at the sequencing stage as a
result of impurities, plasmids were then purified using the commercial kit on the
second attempt.
Once screening of the RE digests confirmed which colony contained a
plasmid with the correct size insert, it was selected and propagated in an LB broth
shake culture, before being purified for sequencing (as in section 2.2.15.iii).
2.2.15.1 Silica method of plasmid purification for screening
From an overnight culture of transformed E. coli DH5α, 1.4 ml was
transferred into a 1.5 ml eppendorf, and pelleted at 6,000 x g for 2 minutes.
Bacterial cell membranes were broken down and chromosomal DNA precipitated
by the addition of lysis buffer, and GTE. The supernatant was removed, and the
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DNA typing of Acanthamoeba sp. 59
remaining pellet resuspended in 120 l GTE (50 mM Glucose, 25 mM Tris pH 8, 10
mM EDTA pH 8). Followed by the addition of 240 µl of lysis buffer (200 mM
NaOH, 1% SDS, (Stored for up to one week at 4C)). The tube was inverted six
times, and incubated on ice for 5 minutes. Then 360 µl KAc (adjusted to pH 5.5
with acetic acid) was added, and the tube inverted six times, followed by
centrifugation at 10,000 x g for 10 minutes. The supernatant was transferred to a
new eppendorf, leaving behind any bacterial cell membranes.
To the new tube 20 µl of fully resuspended silica suspension was added
(2.2.10.2), and the solution vortexed. The now DNA bound silica was pelleted by
centrifugation at 10,000 x g for 10 seconds and all the supernatant removed. The
silica was resuspended in 500 µl of wash solution (50 mM NaCl, 10 mM Tris pH
7.5, 2.5 mM EDTA, 50% ethanol) by gently pipetting. Once washed, the silica was
again pelleted at 10,000 x g for 10 seconds, and the supernatant removed. A second
spin was carried out, and any residual wash buffer removed. The silica was left to
fully dry in their tubes with the caps open, in an incubator at 37C for
approximately 10 minutes. Finally the DNA was eluted into nH2O, by resuspending
the silica in 50 µl nH2O and incubating at 60C for 5 minutes (with occasional
mixing). To remove the silica from the DNA in suspension, the solution was
centrifuged at 12,000 x g for 1 minute, and the supernatant transferred to a new
eppendorf.
Purified plasmids were visualised and quantified in 1.5% agarose gel
electrophoresis (detailed in 2.2.7).
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DNA typing of Acanthamoeba sp. 60
2.2.15.2 Comprehensive plasmid purification for sequencing
From an overnight culture of transformed E. coli DH5α, 1.4 ml was
transferred into a 1.5 ml eppendorf, and pelleted at 12,000 x g for 1 minute, and the
supernatant removed. Bacterial cell membranes were broken down and
chromosomal DNA, precipitated by the addition of lysis buffer, and GTE. The
supernatant was removed, and the remaining pellet resuspended in 200 µl GTE (50
mM Glucose, 25 mM Tris pH 8, 10 mM EDTA pH 8). Followed by the addition of
300 µl of lysis buffer (200 mM NaOH, 1% SDS), (Stored for up to one week at
4C)). The solutions were mixed by tube inversion, and incubated on ice for exactly
5 minutes. A further addition of 300 µl KAc (adjusted to pH 5.5 with acetic acid)
was carried out, and again mixed by inversion, and followed with a 5 minute
incubation on ice. The solutions were then centrifuged at 12,000 x g for 10 minutes,
and the supernatant transferred to a new eppendorf (between 700-750 µl). Rnase A
to a final concentration of 20 µg/ml was added, and the solution left to incubate at
37C for 20 minutes.
To remove unwanted proteins and oligosaccharides two chloroform
extractions were carried out. Here 400 µl of chloroform was added, and the
solutions mixed thoroughly. Centrifuging at 12,000 x g for 1 minute separated the
phases, and the upper aqueous layer removed to a clean eppendorf. Two chloroform
extractions were completed, and then DNA precipitated by adding equal volumes of
isopropanol, and incubating on ice for 10 minutes. All DNA was pelleted by
centrifugation at 12,000 x g for 15 minutes at ambient temperature. The surface of
the pellet was then washed with 500 µl of 70% ethanol. All traces of the alcohol
was removed, following a spin at 12,000 x g for 2 minutes, and air drying at 37C
for approximately 30 minutes. The DNA pellet was dissolved in 32 µl of nH2O.
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DNA typing of Acanthamoeba sp. 61
Once dissolved, 8 µl of NaCl (4 M) and 40 µl of 13% PEG 8,000, was added
and thoroughly mixed, then incubated on ice for at least 20 minutes. The DNA was
again pelleted by spinning at 12,000 x g for 15 minutes at 4C. The supernatant
removed, and the now translucent pellet washed in 70% ethanol, this step was
carried out twice with 12,000 x g centrifugation between each wash. Following the
washing steps all traces of ethanol was removed, after centrifugation at 12,000 x g
for 5 minutes, and the pellet left to air dry at 37C. Ultimately the pellet was
dissolved in 25 µl nH2O.
Purified plasmids were visualised and quantified by 1.5% agarose gel
electrophoresis (detailed in 2.2.7).
2.2.15.3 QIAGEN® plasmid maxi kit
The comprehensive method for purifying plasmids for sequencing was used
initially but replaced by QIAGEN® plasmid maxi kits (QIAGEN Ltd), also based
on alkaline-lysis, and followed by binding of the free DNA to anion-exchange resin,
therefore recovering the plasmids, before dissolving them in nH2O.
Purified plasmids were visualised and quantified by 1.5% agarose gel
electrophoresis (detailed in 2.2.7).
2.2.16 Sequencing
The inserts were commercially sequenced using the standard technique of
automated sequencing at either the Protein Nucleic Acid Chemistry Laboratory
(PNACL; University of Leicester, U.K.) using fluorescently labelled M13 or T7
primers in a 3730 DNA analyser automated sequencer (Applied Biosystems,
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DNA typing of Acanthamoeba sp. 62
Warrington, U.K.), or at Eurofins MWG Operon using their value read sequencing
service.
2.2.17 Sequence analysis
Sequence data was viewed on a chromatogram using Chromas software
version 2.23 (http://www.technelysium.com.au/chromas.html); primer sites were
excluded from analysis to reduce any forced bias. Sequences were then identified
via a basic local alignment search tool (BLAST) (Altschul et al., 1997), using
nucleotide blast (blastn) to locate other highly similar sequences for comparison.
Multiple sequences of the same target region from different strains and species were
aligned together using ClustalW (Larkin M.A. et al., 2007), through the BioEdit
interface (version 7.0.4.1.) (Hall, 1999). With BioEdit, sequences can be aligned
(ClustalW) and extraneous data including primer-binding sites can be easily
removed, which removes any bias caused by their inclusion in the alignment. The
software ClustalW is a general purpose multiple sequence alignment program for
DNA, which aligns the sequences allowing single nucleotide differences to be
identified. Finally phylogenetic and molecular evolutionary analyses were
conducted using MEGA version 5.05 (Kumar et al., 2004), including the
concatenation of the two genes: The multiple aligned sequences were subject to
phylogenetic reconstruction to produce a gene tree using neighbour-joining (NJ)
distance analysis or maximum parsimony (MP) with the kimura two-parameter
correction, and bootstrap support for all phylogenetic trees determined from 1000
replications.
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DNA typing of Acanthamoeba sp. 63
2.3 Results
2.3.1 18S genotyping of strains
Sequences of 18S rDNA and mt cox1/2 were obtained from 28 unique drug
resistant strains, isolated by corneal scrapings from 17 patients. These strains were
added to a range of 12 known isolates, including type species, from all three
morphological groups and 11 T-groups. Sequence variation across ~204 bp and
~564 bp respectively, from the group of 40 isolates was analysed.
Phylogenetic relationships among isolates were examined by maximum
parsimony (MP) and neighbour joining (NJ) analysis, and comparisons were made
of any group’s formed, bootstrap values, and tree topography.
Bootstrap values have been included to help determine groups, as it is
generally considered that values of greater than 70 are evidence to support the
distinction of the clade. Phylogenetic trees produced by both NJ and MP algorithms,
do vary from each other (NJ: <48 = 35%; 49-94 = 50%; >95 = 15%. MP: <48 =
86%; 49-94 = 11%; >95 = 3%), with NJ trees resulting in a greater number of
higher bootstrap values. However the clusters of taxa remain largely the same,
although their position within the tree vary depending on the analysis used.
The 18S NJ tree (Figure 1) shows multiple species are clumped together in
genotype groups T3, T4 and T11, with the maximum pairwise distance value
between the three clades at 0.055 (T11 verses T3), and a minimum value of 0.033
(T3 verses T4 (Table 4)). The 18S tree highlights the close relationship between T3,
T4 and T11 clades. With the T11 group located between two subsections of T3,
where isolates AK 1a, AK 4, AK 6 and AK 14 are above the T11 cluster, and AK 5,
AK 5a, AK 11 and A. griffinii 1501/4 are below (Figure 1). Also located a separate
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DNA typing of Acanthamoeba sp. 64
branch of its own is the T4 isolate A. castellanii 1501/1a, which when analysed here
clusters above T11 and below the upper T3 subsection.
Table 4. Estimates of evolutionary divergence over 18S sequence pairs between T-
group genotypes. The number of base substitutions per site from between sequences
are shown. Analyses were conducted using the Maximum Composite Likelihood
model (Tamura et al., 2004). The analysis involved 40 nucleotide sequences. All
positions containing gaps and missing data were eliminated. There were a total of
336 positions in the final dataset. Evolutionary analyses were conducted in MEGA5
(Tamura et al., 2011) in press.
T11 T3 T4 T6 T2 T10 T9 T8 T5
T3 0.055 - - - - - - - -
T4 0.034 0.033 - - - - - - -
T6 0.104 0.081 0.084 - - - - - -
T2 0.123 0.109 0.111 0.034 - - - - -
T10 0.101 0.118 0.093 0.14 0.161 - - - -
T9 0.221 0.236 0.219 0.276 0.29 0.229 - - -
T8 0.258 0.211 0.229 0.263 0.274 0.25 0.118 - -
T5 0.091 0.091 0.072 0.079 0.07 0.1 0.229 0.249 -
T7 0.256 0.199 0.226 0.238 0.271 0.272 0.109 0.07 0.248
When comparing average pairwise distances of all the T-groups, maximum
values occur between T2 verses T8 (0.274), and minimum between T3 and T4
(0.033) (Table 4). Differences between the closely related sequence types; T3, T4
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DNA typing of Acanthamoeba sp. 65
and T11 in this study, are less than the previously recorded value of at least 5%
(which is an arbitrary value) (Stothard et al., 1998). Here, dissimilarity values of
3.3% (T4 verses T3), 5.5% (T3 verses T11) and 3.4% (T4 verses T11) were
obtained. Sequences types T2 and T6 also showed high sequence similarity, with a
dissimilarity score of 3.4%. Differences between genotypes were always greater
than within sequence types.
Of the 28 clinical AK isolates, 11 strains collected from eight patients were
analysed and assigned to the T4 clade (Figure 1). While seven AK isolates from six
patients were assigned to the clade T3 with A. griffinii 1501/4. The final 11 isolates
from six patients were included on three branches within T11 with A. hatchetti Bh2.
Distinct sequence types placed at the very bottom of the tree are all
morphological group I species, which have previously been assigned to T7 (A.
astronyxis 30137), T8 (A. tubiashi 30876), and T9 (A. comandoni 1501/5). Placed
between the lower morphological group I species, are the closely linked T2 and T6
species (A. palestinensis 1501/3c and A. palestinensis 1547/1), along with T9 A.
lenticulata PD2S on a separate branch.
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DNA typing of Acanthamoeba sp. 66
AK 17
A. polyphaga Ros
AK 15
AK 13
AK 12a
AK 12
AK 7a
AK 6a
AK 7
AK 9
AK 9a
AK 18
T4
AK 1a
AK 4
AK 6
AK 14
T3
T4 A castellani 1501/1a
AK 10a
AK 19
AK 10
AK 1
AK 8a
AK 3
AK 16
AK 16a
AK 3i
AK 3a
AK 3b
A. hatchetti Bh2
T11
AK 5
AK 5a
AK 11
A. griffinii 1501/4
T3
T5 A. lenticulata PD2S
T6 A. palestinensis 1547/1
T2 A. palestinensis 1501/3c
T10 A. culbertsoni 30171
T9 A. comandoni 1501/5
T8 A. tubiashi 30876
T7 A. astronyxis 3013790
100
88
76
98
95
70
76
45
64
60
68
42
32
43
24
22
50
37
61
0.02
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DNA typing of Acanthamoeba sp. 67
Figure 1. Neighbour joining distance tree based on partial 18S rDNA sequences of
Acanthamoeba spp. The tree is unrooted and obtained by Kimura two-parameters
correction for multiple substitutions using MEGA (5.05). The tree is based on
reference bp from 1,175 to 1,379. The scale bar represents the corrected number of
nucleotide substitutions per base using Kimura method. Designated T-groups are
shown.
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DNA typing of Acanthamoeba sp. 68
T6 A. palestinensis 1547/1
T2 A. palestinensis 1501/3c
T3 AK 14
T3 AK 6
T3 AK 4
T3 AK 1a
AK 9a
AK 9
AK 18
T4
AK 15
AK 17
AK 13
AK 12
AK 12a
AK 7
AK 6a
A. polyphaga Ros
T4
T4 AK 7a
A. griffinii 1501/4
AK 11
AK 5
AK 5a
T3
T4 A castellani 1501/1a
AK 10
AK 19
AK 10a
AK 1
AK 8a
AK 3b
AK 16a
AK 3i
A. hatchetti Bh2
AK 3
AK 16
AK 3a
T11
T5 A. lenticulata PD2S
T10 A. culbertsoni 30171
T9 A. comandoni 1501/5
T8 A. tubiashi 30876
T7 A. astronyxis 30137
34
96
15
21
72
15
28
3
2
1
2
5
23
49
1
27
17
2
0
0
0
0
1
3
69
1
2
13
12
1
0
1
2
14
36
99
56
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DNA typing of Acanthamoeba sp. 69
Figure 2. Maximum parsimony distance tree based on partial 18S rDNA sequences
of Acanthamoeba spp. The tree is unrooted and obtained by Kimura two-parameters
correction for multiple substitutions using MEGA (5.05). The tree is based on
reference bp from 1,175 to 1,379. Designated T-groups are shown.
2.3.2 Typing strains with cox1/2 gene
Sequences of mt cox1/2 were obtained from 28 unique drug resistant strains,
isolated by corneal scrapings, from 17 patients. These strains were added to a range
of 12 known isolates, including type species, from all three morphological groups
and 11 T-groups. Sequence variation across ~564 bp, from the group of 40 isolates
was analysed.
Phylogenetic relationships among isolates were examined by maximum
parsimony (MP) (Figure 3) and NJ analyses (Figure 4).
When examined by NJ analysis as with cox1/2, A. tubiashi 30876 (T8) was
found to be distinct from the main group of Acanthamoeba sp., but interestingly
even more distinct was the species A. hatchetti Bh2 (Figure 4). By removing these
two distinct species from the comparison, the branches of the tree become more
elongated (data not shown). Unfortunately, despite continued efforts, mt cox1/2
sequences could not be obtained for Hartmannella or Balamuthia spp. in order that
they be included as an outlying species or true evolutionary ancestor, and allow A.
tubiashi and A. hatchetti to remain within the main section of the tree.
Figure 3, displays phylogenetic relationships when examined by maximum
parsimony and includes bootstrap values to help determine clades, as it is generally
considered that values of greater than 70 are evidence to support the distinction of
the clade. Phylogenetic trees produced by both NJ and MP algorithms, do not vary
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DNA typing of Acanthamoeba sp. 70
extensively from each other (NJ: <48 = 32%; 49-94 = 42%; >95 = 19%. MP: <48 =
50%; 49-94 = 29%; >95 = 18%), and the clusters of taxa remain the same, although
their position within the tree may vary depending on the analysis used. The outlying
distinct taxa change, from A. tubiashi 30876 (T8) and A. hatchetti Bh2 (T11) with
NJ, to group I species A. astronyxis 1534/1 with MP analysis.
Using cox1/2 tree analysis with bootstrap values, eight groups can be
identified (Figure 3 and 4) (Group A-H). Across these sequence types, the
pathogenic strains including the Moorfield AK strains, are resolved into smaller
clades, than the general clumping together formed by 18S T-group analysis. T11
isolates are no longer clustered together, especially relevant to the repeat isolates
from Patient 3 (AK 3 AK 3i, AK 3a, AK 3b, AK 16, and AK 16a), which were all
located in a tight clade by 18S (Figure 1 and 2), but are now distributed throughout
cox1/2 groups A, C, E, G and H. Within this study A. castellanii 1501/1a, AK 10a,
A. palestinensis 1547/1, A. griffini 1501/4, A. tubiashi 30876, A. hatchetti Bh2, A.
astronyxis 1534/1, A. comandoni 1501/5 and A. palestinensis 1501/3c cannot be
assigned to a clade with a relevant bootstrap value of over 70.
Strains with pathogenic potential are found throughout the tree, and AK and
encephalitis causing strains are not distinct from those which are thought be non-
pathogenic. Interestingly, cox1/2 groups were not limited to repeat isolate sequences
but often contain isolates collected from different patients/locations. The range of
pairwise distance values between the cox1/2 sequence groups varies from a
minimum of 0.036 (group B verses C), to a maximum of 0.286 (group H verses F)
(Table 5).
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DNA typing of Acanthamoeba sp. 71
Distinct species at the base of the cox1/2 trees differs depending on analysis.
By NJ A. hatchetti Bh2 and A. tubiashi 30876 are the distinct strains, but by MP
analysis the outlying species is A. astronyxis 1534/1. Sequence similarity between
the cox1/2 groups is always higher than 5%, and the differences between sequence
types is always greater than within sequence types.
Table 5. Estimates of evolutionary divergence over cox1/2 sequence pairs between
groups (A-G). The number of base substitutions per site from between sequences
are shown. Analyses were conducted using the Maximum Composite Likelihood
model (Tamura et al., 2004). The analysis involved 40 nucleotide sequences. All
positions containing gaps and missing data were eliminated. There were a total of
336 positions in the final dataset. Evolutionary analyses were conducted in MEGA5
(Tamura et al., 2011) in press.
G C E H B A D
C 0.229 - - - - - -
E 0.23 0.101 - - - - -
H 0.23 0.228 0.224 - - - -
B 0.189 0.036 0.09 0.193 - - -
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DNA typing of Acanthamoeba sp. 72
A 0.219 0.039 0.092 0.233 0.023 - -
D 0.242 0.095 0.105 0.247 0.07 0.085 -
F 0.265 0.201 0.222 0.286 0.172 0.207 0.237
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DNA typing of Acanthamoeba sp. 73
AK 12 (T4)
AK 12a (T4)
AK 3a (T11)
AK 19 (T11)
AK 3i (T11)
A. polyphaga Ros (T4)
Gp A
AK 9 (T4)
AK 9a (T4)
AK 15 (T4)
AK 17 (T4)
Gp B
AK 10 (T11)
AK 1a (T3)
AK 6 (T3)
AK 6a (T4)
AK 13 (T4)
Gp C
A. palestinensis 1547/1 (T6)
AK 14 (T3)
AK 4 (T3)Gp D
AK 7 (T4)
AK 7a (T4)
AK 16 (T11)
AK 18 (T4)
Gp E
A. castellani 1501/1A (T4)
AK 10a (T11)
A. culbertsoni 30171 (T10)
A. lenticulata PD2S (T5)Gp F
A. comandoni 1501/5 (T9)
A. palestinensis 1501/3c (T2)
A. hatchetti Bh2 (T11)
A. tubiashi 30876 (T8)
AK 8a (T4)
AK 1 (T11)
AK 16a (T11)
Gp H
A. griffinii 1501/4 (T3)
AK 5 (T3)
AK 11 (T3)
AK 3b (T11)
AK 5a (T3)
AK 3 (T11)
Gp G
Gp G A. astronyxis 1534/1 (T7)
84
93
12
100
81
63
98
99
26
66
60
100
44
10
4
11
87
41
78
32
100
14
61
15
2
16
21
89
96
35
17
24
66
21
14
66
100
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DNA typing of Acanthamoeba sp. 74
Figure 3. Maximum parsimony tree based on partial mt cox1/2 sequences of
Acanthamoeba spp, with cox1/2 groups (A-H) and T-groups (T2-T11) included. The
tree is unrooted and obtained by Kimura two-parameters correction for multiple
substitutions using MEGA (5.05). The tree is based on reference bp 8,002 to 8,566.
Bootstrap values have been included, based on 1,000 bootstrap values, and are
placed at the nodes they apply to.
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DNA typing of Acanthamoeba sp. 75
AK 3i (T11)
AK 3a (T11)
AK 19 (T11)
A. polyphaga Ros (T4)
AK 12 (T4)
AK 12a (T4)
Gp A
AK 9 (T4)
AK 9a (T4)
AK 15 (T4)
AK 17 (T4)
Gp B
AK 10 (T11)
AK 6 (T3)
AK 6a (T4)
AK 1a (T3)
AK 13 (T4)
Gp C
A. palestinensis 1547/1 (T6)
AK 14 (T3)
AK 4 (T3)Gp D
AK 7 (T4)
AK 7a (T4)
AK 16 (T11)
AK 18 (T4)
Gp E
AK 10a (T11)
A. castellani 1501/1A (T4)
A. comandoni 1501/5 (T9)
A. culbertsoni 30171 (T10)
A. lenticulata PD2S (T5)Gp F
A. palestinensis 1501/3c (T2)
A. griffinii 1501/4 (T3)
A. astronyxis 1534/1 (T7)
AK 5 (T3)
AK 11 (T3)
AK 5a (T3)
AK 3 (T11)
AK 3b (T11)
Gp G
AK 1 (T11)
AK 8a (T4)
AK 16a (T11)
Gp H
A. tubiashi 30876 (T8)
A. hatchetti Bh2 (T11)
99
99
62
79
74
96
74
94
85
80
93
57
99
12
14
11
21
22
98
39
90
31
15
85
96
5633
2566
97
63
0.05
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DNA typing of Acanthamoeba sp. 76
Figure 4. Neighbour-joining distance tree based on partial mt cox1/2 sequences of
Acanthamoeba spp with cox1/2 groups (A-H) and T-groups (T2-T11) included. The
tree is unrooted and obtained by Kimura two-parameters correction for multiple
substitutions using MEGA (5.05). The tree is based on reference bp 8,002 to 8,566.
The scale bar represents the corrected number of nucleotide substitutions per base
using Kimura method.
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DNA typing of Acanthamoeba sp. 77
2.3.3 Introns, multiple alleles and mixed infections
Using repeat patient isolates evidence presented here suggests two patients
contained species with multiple alleles.
Repeat samples include AK 1 and AK 1a from Patient one, AK 6 and AK 6a
from Patient six, and AK 3, AK 3i, AK 3a, AK 3b, AK 16 and AK 16a from Patient
3. 18S NJ and MP tree analysis shows Patient one’s isolate AK 1 in T11 clustering
independently from the second isolate, AK 1a in T3. The T3 clade also contains an
isolate from Patient six (AK 6a). While the other Patient six isolate, AK 6 clusters
within T4 (Figure 1 and 2).
However by sequencing a second gene, cox1/2, Patient six’s isolates AK 6 and
AK 6a, are found to have sequence homology with a pairwise distance score of 0
(cox1/2 group c by NJ and MP trees) (Figure 3 and 4), there by indicating multiple
alleles of 18S but not cox1/2.
This was not so for Patient one’s isolates (AK 1 and AK 1a), which showed
sequence variation occurring in both cox1/2 and 18S genes. The high dissimilarity
sequence variation of cox1/2 10.2% (pairwise value of 0.102) and 18S (pairwise
value 0.052) genes, suggests the presence of a mixed infection (Figure 1, 2, 3 and
4).
Isolates AK 3, AK 3i, AK 3a, AK 3b, AK 16 and AK 16a are all repeat strains
collected from Patient three. When aligned by 18S they are 100% identical, and
cluster tightly together on the same branch within T11, with A. hatchetti Bh2
(Figure 1 and 2). However by cox1/2, analysis of the data suggests that there are
four copies of the gene within the strain. The isolates are spread out across the tree
in five groups, A, C, E, G and H (Figure 3 and 4), with sequence dissimilarity
values ranging from 4.6 to 10.2% (Table 6).
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DNA typing of Acanthamoeba sp. 78
Within the region of the sequenced region of the cox1/2 gene, analysis
shows no indication of introns, while evidence for the presence of multiple alleles
and mixed infections were found.
Table 6. Pairwise distance values between cox1/2 sequences of repeat isolates from
Patient three. The number of base substitutions per site from between sequences are
shown. Analyses were conducted using the Maximum Composite Likelihood model
(Tamura et al., 2004). The analysis involved 40 nucleotide sequences. All positions
containing gaps and missing data were eliminated. There were a total of 336
positions in the final dataset. Evolutionary analyses were conducted in MEGA5
(Tamura et al., 2011) in press.
AK 3 AK 16 AK 16a AK 3i AK 3a
AK 16 0.098 - - - -
AK 16a 0.098 0.100 - - -
AK 3i 0.094 0.046 0.102 - -
AK 3a 0.094 0.046 0.102 0.000 -
AK 3b 0.000 0.098 0.098 0.098 0.094
2.3.4 Concatenated sequence data
Trees were also analysed from 18S and cox1/2 data, which had been
concatenated together to determine which resulting tree from all three data sets
provided the greatest resolution.
MP and NJ trees both contained 37 nodes supported by bootstrap values.
The tree from NJ analysis (Figure 6) contains more bootstrap values of greater than
49 compared with MP trees (Figure 5) (NJ- <48 = 24%, 49-94 = 46%, >95 = 32%;
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DNA typing of Acanthamoeba sp. 79
MP- <48 = 32%, 49-94 = 46%, >95 = 16%). Both contain similar clade formation,
with differing distinct strains at the base of the trees, following trees derived from
cox1/2 data alone. The distinct species by NJ are A. tubiashi 30876, and A.
hatchetti, which by MP form their own group between cox1/2 groups E and H.
Leaving A. astronyxis 1534/1 to be the distinct species in concatenated MP trees.
Another distinction between MP and NJ trees with concatenated data is the split of
group B, with AK 15 and AK 17 in the top half, and AK 9 and AK 9a in the second
half (Figure 5).
Clade formation of concatenated trees most resembles those of cox1/2, rather
than 18S. However shorter branch lengths are found on the concatenated tree than
those of cox1/2. The concatenated tree provides more resolution between species,
rather than the clumping found with 18S trees, supported by a greater number of
nodes with significant bootstrap values of greater than >95.
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DNA typing of Acanthamoeba sp. 80
AK 3i (T11)
AK 19 (T11)
AK 3a (T11)
AK 12 (T4)
AK 12a (T4)
A. polyphaga Ros (T4)
Gp A
AK 15 (T4)
AK 17 (T4)Gp B
AK 9 (T4)
AK 9a (T4)Gp B
AK 10 (T11)
AK 1a (T3)
AK 6 (T3)
AK 6a (T4)
AK 13 (T4)
Gp C
AK 4 (T3)
AK 14 (T3)Gp D
A. palestinensis 1547/1 (T6)
AK 7 (T4)
AK 7a (T4)
AK 16 (T11)
AK 18 (T4)
Gp E
A. castellani 1501/1A (T4)
A. hatchetti Bh2 (T11)
A. tubiashi 30876 (T8)
AK 16a (T11)
AK 1 (T11)
AK 8a (T11)
Gp H
AK 10a (T11)
A. palestinensis 1501/3c (T2)
A.culbertsoni 30171 (T10)
A lenticulata PD2S (T5)Gp F
A. comandoni 1501/5 (T9)
A. griffinii 1501/4 (T3)
AK 5 (T3)
AK 11 (T3)
AK 5a (T3)
AK 3 (T11)
AK 3b (T11)
Gp G
Gp G A. astronyxis 1534/1 (T7)
67
82
99
99
99
30
99
93
83
68
72
86
89
56
99
60
54
98
17
34
50
41
65
20
68
13
78
21
13
31
60
11
5
22
27
70
89
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DNA typing of Acanthamoeba sp. 81
Figure 5. Maximum parsimony distance tree based on concatenated partial mt
cox1/2 with 18S sequences of Acanthamoeba spp with cox1/2 groups (A-H) and T-
groups (T2-T11) included. The tree is unrooted and obtained by Kimura two-
parameters correction for multiple substitutions using MEGA (5.05). The tree is
based on reference bp 8,002 to 8,566.
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DNA typing of Acanthamoeba sp. 82
AK 12 (T4)
AK 12a (T4)
A. polyphaga Ros (T4)
AK 19 (T11)
AK 3i (T11)
AK 3a (T11)
Gp A
AK 9 (T4)
AK 9a (T4)
AK 15 (T4)
AK 17 (T4)
Gp B
AK 10 (T11)
AK 1a (T3)
AK 6 (T3)
AK 6a (T4)
AK 13 (T4)
Gp C
A. palestinensis 1547/1 (T6)
AK 4 (T3)
AK 14 (T3)Gp D
AK 16 (T11)
AK 18 (T4)
AK 7 (T4)
AK 7a (T4)
Gp E
AK 10a (T11)
A. castellani 1501/1A (T4)
A. palestinensis 1501/3c (T2)
A.culbertsoni 30171 (T10)
A lenticulata PD2S (T5)Gp F
A. comandoni 1501/5 (T9)
A. griffinii 1501/4 (T3)
AK 3 (T11)
AK 3b (T11)
AK 5 (T3)
A. astronyxis 1534/1 (T7)
AK 5a (T3)
AK 11 (T3)
Gp G
AK 16a (T11)
AK 1 (T11)
AK 8a (T11)
Gp H
A. tubiashi 30876 (T8)
A. hatchetti Bh2 (T11)
100
77
100
99
45
37
79
87
100
74
62
97
64
99
9932
32
49
30
36
99
54
99
96
97
85
94
35
52
4479
3396
77
75
72
88
0.1
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DNA typing of Acanthamoeba sp. 83
Figure 6. Neighbour-joining distance tree based on concatenated partial mt cox1/2
with 18S sequences of Acanthamoeba spp with cox1/2 groups (A-H) and T-groups
(T2-T11) included. The tree is unrooted and obtained by Kimura two-parameters
correction for multiple substitutions using MEGA (5.05). The tree is based on
reference bp 8,002 to 8,566. The scale bar represents the corrected number of
nucleotide substitutions per base using Kimura method.
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DNA typing of Acanthamoeba sp. 84
2.4 Discussion
2.4.1 Genotyping Acanthamoeba sp. with 18S sequences
There are many examples of the use of 18S based molecular studies for
epidemiological typing, to uncover relationships between patients with
Acanthamoeba infections and their environments (Maghsood et al., 2005, Schroeder
et al., 2001, Walochnik et al., 2000). As all forms of life share features of structure
and sequence within rRNA, analysis of it has been used to map phylogenetic history
across organisms at all levels, including the discovery of a third kingdom the
archaea, in addition to prokaryotes and eukaryotes (Woese et al., 1990).
Within Acanthamoeba, rRNA genes show a relatively slow rate of change,
and are a suitable candidate to base a genotyping system on (Gast et al., 1996).
From the similarities of the genotype clusters found by analysis of both nuclear
(Rns) and mitochondrial (rns) small subunit rRNA genes, it can be assumed that
phylogenetic clades based on 18S sequences represent evolutionary history (Ledee
et al., 2003). Thereby inferring all clades linked with AK (T2, T3, T4, T5, T6 and
T11), and GAE (T1, T4, T10 and T12), each share evolutionary adaptations to have
the potential to cause infections. In general T4 genotypes are responsible for most
AK infections, such a marked phylogenetic localisation suggests either, innate
pathogenic potential, a peculiarity of the gene sequence, or increased prevalence in
the environment (Ledee et al., 2003). Yet evidence has been presented to show the
close relationship between an eye and a lung isolate when examined by 18S
sequence, leading the authors to conclude that any pathogenic strain may be capable
of infecting more than one type of tissue (Gast et al., 1996).
By sequencing the 18S gene of Acanthamoeba it has been shown that most
isolates, both environmental and clinical including those from all forms of
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DNA typing of Acanthamoeba sp. 85
acanthamebiasis can be typed into the T4 genotype (Gast et al., 1996, Schroeder et
al., 2001, Stothard et al., 1999, Stothard et al., 1998). In a single study examining
249 isolates, 179 of them belonged to T4. These isolates were obtained from both
environmental and clinical sources from, AK patients and non-AK including CSF,
brain, skin and lungs (Booton et al., 2005).
Isolates of the T4 genotype appear to be the predominant cause of AK and
GAE, and the only genotypes yet to be linked to human disease are T7, T8, T9, T13,
T14 and T15 (Khan, 2009). To find pathogenic strains classified to groups other
than T4 is less common (Booton et al., 2005, Gast et al., 1996, Stothard et al., 1999,
Stothard et al., 1998). However, exception has been shown in this study, with many
of the isolates belonging to genotypes T3, T4 and T11, but as this study group
contains largely AK causing strains it presents a strong bias towards pathogenic
genotypes. From the clinical isolates collected from 17 patients, eight of the patients
had isolates belonging to T4 (11 strains); six patients had isolates belonged to T3
(seven strains), and five patients were infected with T11 isolates (11 strains). Eleven
of the possible 15 T-groups were included in this study, and of these, three belonged
to non-pathogenic genotypes: As expected they clustered close to each other at the
bottom of the tree, distinct from the main taxa, and formed clades T7, T8 and T9.
Despite tree topology mainly following genotypes, with the exception of the split
T3 group average sequence divergence between the genotypic clades T3, T4 and
T11 based on pairwise sequence comparison are lower than the current arbitrary cut
off value of 5% (T3-T4: 3.3%; T4-T11: 3.4%), (Gast et al., 1996). The close
association between isolates from the three genotypes (T3/4/11) shown by pairwise
sequence comparisons may explain why the in both MP and NJ trees T3 isolates are
split into smaller clusters.
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DNA typing of Acanthamoeba sp. 86
Although the 18S typing system has been used here to effectively classify a
unique group of AK causing isolates, and consequently confirm the robustness of
the current system. As expected, the system was not effective at resolving the
species and provided little definition between them. Instead multiple isolates were
clumped into a few large groups. A proposal has been put forward to rename
Acanthamoeba strains by T-group rather than species name, saving confusion
caused as a result of the historical classification system (Stothard et al., 1998).
However given the clustering of many species into few clades by the T-group
system, such renaming would most likely cause even more confusion to an already
complicated taxonomic system. Especially given the close association between
certain clades, where T2 and T6 have recently been referred to within the literature
as T2-T6 clade (Corsaro and Venditti, 2010, Huang and Hsu, 2010, Niyyati et al.,
2009).
Even though partial 18S sequences were obtained from all study isolates,
once subjected to phylogenetic analysis, distance trees with comparable topology to
previous studies were produced. Amplification of a shorter sequence both increases
the throughput of isolates, and reduces the potential for taq errors, without the loss
of vital data for analysis.
With the recent increase in AK incidence, and the latest outbreak in the
USA, more and more cases are being diagnosed. Ultimately this is leading to an
increase in the development of resistance to drug treatments. Highlighting the need
for an improved system with a greater power of differentiating than the current T-
group genotyping system.
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DNA typing of Acanthamoeba sp. 87
2.4.2 Genotyping based on cox1/2 sequences
Although rRNA genes prove the most attractive and popular choice for
intraspecific phylogeny and taxonomy studies, mitochondrial genes are also suitable
for use as a resolving tool to better understand close phylogenetic relationships
because they evolve approximately 10 fold faster than nuclear genes (Brown et al.,
1982). The mitochondrial genome is most likely to be under different constraints, as
it is located within a cell organelle rather than the nucleus (Ledee et al., 2003). The
Acanthamoeba mitochondrial genome appears to be conserved and have evolved
relatively slowly (Burger et al., 1995).
An earlier study has looked at sequence variation of cytochrome oxidase
genes within Acanthamoeba species, revealing a large degree of nucleotide
variation, enough to differentiate eight AK patient isolates, as well as matching six
of them by sequence homology to their respective tap water isolates (Kilvington et
al., 2004). Here the same mitochondrial gene cox1/2 was trialled as a suitable
contender for an alternative/additional target for genotyping Acanthamoeba species
rather than the current 18S T-group system.
The high degree of nucleotide variation within cox1/2 sequences of
Acanthamoeba isolates made the search for suitable primers difficult. The primers
were required to be Acanthamoeba specific, and suitable for use with as many
strains as possible. A second objective was to design a pair of primers that produced
a sequence product ideally <1,000 bp, but still include a region of high variability.
The resulting primer pair CoxA-486F and CoxA-1057R is sensitive and highly
effective, with the ability to work well with a range of unusual strains. Even
amplifying the cox1/2 gene of an atypical AK amoeba AK95/1153, where the 18S
Acanthamoeba specific primers JDP1F and JDP2R failed, and only the universal
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DNA typing of Acanthamoeba sp. 88
primers succeeded (18sF: 18sR). Sequence analysis of the strain AK95/1153 has
provided surprising results with its cytochrome oxidase sequence matching
Acanthamoeba, but its 18S sharing 96% homology with F. aegytia. Further research
is needed to understand more of its genetics and evolution, but initial observations
have revealed some binucleate cysts, further linking it to the Flamella genus. If
confirmed as a Flamella sp., this would be first time the genus had been associated
with a case of human keratitis.
Evidence has been presented here to indicate that an alternative system that
has the ability to resolve Acanthamoeba isolates with pathogenic potential has been
developed. Genotyping by cox1/2 sequences does not group multiple pathogenic
strains into a few clades, as does the T-group system. However, the majority of
strains included in this study are AK causing isolates, the addition of environmental
strains would show their effect on the branching patterns, and in turn highlight the
robustness of the system.
2.4.3 Typing with concatenated gene sequences
Recent studies have shown multiple housekeeping genes can be
concatenated to form one super-gene alignment for building more robust
phylogenetic trees, which provide better discrimination power (Devulder et al.,
2005, Gadagkar et al., 2005, Kurtzman and Robnett, 2007).
This concatenated approach within the Acanthamoeba genus using the well
documented SSU 18S rDNA gene from the T group system, in combination with
another housekeeping gene, the mitochondrial cox1/2, has resulted in trees with
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DNA typing of Acanthamoeba sp. 89
greater powers of discrimination. Which ultimately improve the ability to
phylogenetically classify the subgenus and improve the diagnosis of AK.
The phylogenetic trees based on concatenated data trees bears most
resemblance to the trees based on cox1/2 data, and increases the discrimination
power of the phylogenetic analyses within Acanthamoeba spp., something that has
not yet been achieved by the less discriminatory T-group system.
Results here do not contradict current opinions, now strongly biased towards
using a multigene approach to increase the resolution power of identifying species
(Devulder et al., 2005, Gadagkar et al., 2005, Kurtzman and Robnett, 2007).
However, although the concatenated tree has more discriminatory capabilities than
those with 18S data alone, little improvement is gained over using cox1/2 data
independently. The addition of even more genes in a concatenated dataset should be
tested to ascertain if further discriminatory power would be obtained. Further
analysis should also be considered using a larger data set than studied here.
2.4.4 Multiple alleles and mixed infections using 18S and cox1/2
Historically, evidence has suggested the presence of a single allele of the
18S gene occurring within Acanthamoeba, despite estimates suggesting that A.
castellanii Neff has several hundred copies of the gene. More recent evidence has
shown multiple alleles are actually present in at least seven isolates (Stothard et al.,
1998). For the presence of multiple alleles of 18S to be due to errors by taq
polymerase, the same allelic-specific mistakes would have to occur in two groups of
isolates, which is unlikely. However multiple alleles can occur as a result of
genetically different strains being present within the initial amoebae cultures.
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The discovery of multiple alleles can be a benefit, and aid a better
understanding of the course of the infection.
Using a repeat series of isolates collected from a single patient, a second
study determined if drug resistance to propamidine had developed, or if the failure
of the patient to respond to treatment was due to a mixed infection (Ledee et al.,
1998). The presence of identical 18S multiple alleles in both drug-sensitive and
drug-resistant isolates (pre and post treatment), led to the conclusion that resistance
had developed throughout the cause of the treatment.
This study has shown the 18S gene sequence from two strains isolated from
Patient 6 (AK 6 and AK 6a), cluster independently on two branches, each in a
different clade, AK 6a in T4 and AK 6 in T3. However when analysed by cox1/2
sequence, both strains show sequence homology, and are found together in cox1/2
group c. Suggesting Patient 6 was infected with a strain of Acanthamoeba which has
multiple alleles of it’s 18S gene. Unlike here, the previous study which uncovered
multiple alleles of 18S in seven isolates, found where they do occur, both alleles fall
within the same 18S rDNA sequence type, which as a consequence is unlikely to
have a major impact on genotyping (Stothard et al., 1998).
Analyses of 18S sequences from repeat isolates of Patient one (AK 1 and AK 1a)
showed the presence of two sequence types (cox1/2 c and h), with AK 1 located in
T11, and AK 1a in T3. However, analysis of the cox1/2 gene from both isolates
shows a similar pattern, with two distinct sequence types. Evidence no longer points
to multiple alleles in either genes, but instead a mixed infection within the one
patient.
Evidence for the presence of multiple alleles second gene, cox1/2, was found
in this study. Six isolates recovered from a single patient (three), collected as repeat
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samples, demonstrated four sequence types (cox1/2 a, e, g, and h), while they shared
sequence homology when analysed by 18S. Multiple alleles have also been
discovered in the Acanthamoeba lactic dehydrogenase gene (Ward and al-Abidin,
1988), but no others as yet have been identified. This is likely to change with the
imminent release of the complete high-quality sequencing of A. castellanii Neff
genome, which is currently in progress.
The use of two genes in relatively quick, and robust (in the case of 18S, and
yet to be proven in cox1/2) PCR can rapidly confirm or rule out the possibility of
mixed infections within a patient. Early detection of a mixed infection would
benefit the patient by improving prognosis, and reducing the risk of drug resistance
developing. In addition, a genotyping system capable of identifying multiple alleles
allows drug resistance to be tracked throughout the course of the treatment.
2.4.5 Conclusion
Speciation of the amoebae involved in acanthamebiasis may have
implications regarding epidemiology and the course of the infection, but is unlikely
to have a huge effect on the treatment, while the discovery of a mixed infection in
an individual may. The combined use of two systems to genotype can rapidly
determine the occurrence of a mixed infection, where a single system would
struggle.
Evidence here, supported previous studies (Stothard et al., 1998) identifying
multiple alleles of 18S occurring within Acanthamoeba, and additionally recognised
multiple alleles of the cox1/2 gene present within the genus. Tracking multiples
alleles of both genes and using them as epidemiological markers can track drug
regime resistance.
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The T-group system is proving an invaluable tool within epidemiological
studies, and is continuing to evolve with the addition of new genotypes. However
the T-group system does not effectively resolve the isolates with pathogenic
potential, and clumps large numbers of strains within groups T3, T4 and T11. The
search for an efficient, fast and robust system to differentiate between virulent and
non-virulent strains has been an ongoing concern. This study has presented cox1/2
as a suitable contender to 18S for use in a phylogenetic system in the
epidemiological typing of Acanthamoeba, as it provides much more distinction
between the strains than the T-group system. However, the combined use of both
systems in this study led to the discovery of the mixed infection in Patient one. This
had the potential to have been interpreted as multiple alleles had either system been
used independently. Leading ultimately to the conclusion that the use of both
systems to genotype Acanthamoeba is synergistic.
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3 SWEDISH GAE STUDY
3.1 Introduction
Species of Acanthamoeba are one of the causative agents of granulomatous
amoebic encephalitis also known as GAE. It is a rare disease, with less than 200
cases caused by Acanthamoeba documented within the literature (Schuster and
Visvesvara, 2004). The skin and the olfactory neuroepithelium are the most likely
route of infection (Walochnik et al., 2008). Once inside the body, the amoebae most
likely gain access into the CSF by hematogenous, or by passing directly through the
neuroepithelium (Marciano-Cabral, 2003). Typically the infections occur in the
brain and/or the lungs of severely debilitated or immune suppressed individuals.
The infection presents with hemorrhagic necrotising lesions, generally found in the
cerebrum, cerebellum and brain stem, with symptoms including headaches, fever,
behavioural abnormalities and hemiparesis (Marciano-Cabral, 2003, Martinez and
Janitschke, 1985, Schuster and Visvesvara, 2004, Walochnik et al., 2008).
Diagnosis is almost always made post-mortem following brain tissue
biopsies, and indirect immunofluorescence (IIF) staining of tissue sections (Bloch
and Schuster, 2005, Schuster and Visvesvara, 2004). However pre-mortem
diagnosis of GAE has been reported within the literature. Achieved as a result of
positive Acanthamoeba-specific PCR of several biopsy tissue and fluid specimens,
including cerebrospinal fluid (CSF), bronchoalveolar lavage specimens (BAL),
skin, lung, and brain tissue (from the main lesion). All of which had been
Acanthamoeba culture negative when tested (Walochnik et al., 2008).
On a rare occasion, direct recovery of Acanthamoeba from CSF has been reported
(Callicott, 1968, Sharma et al., 1993, Singhal et al., 2001), however amoebae are
generally not found in the CSF.
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PCR has proved to be an invaluable tool, used to diagnose Acanthamoeba
infections. The highly sensitive T-group typing system has also been used to
determine epidemiological association between a keratitis-causing strain of
Acanthamoeba, the patient, their contact lens storage case and their domestic water
supply (Ledee et al., 1996). An alternative system based on mt DNA profiles,
comparing a region of the cox1/2 gene, has also been used to link six patients and
their home tap water isolates (Kilvington et al., 2004).
In an unprecedented case our laboratory received repeat clinical and water
samples from three patients from a single ward within a Swedish hospital. The
patients were immune compromised paediatrics with severe debilitating illnesses.
Extraordinarily, they had all contracted fatal GAE whilst being treated on the same
intensive care unit (ICU) within the hospital. The sample set included repeat CSF
samples from all three patients and hospital water samples from four shower rooms,
a pool, and the main in-water supply to the ward. Such an outbreak of
Acanthamoeba GAE infections has never before been documented, as occurrences
of cases previously have been sporadic involving individuals in isolated cases
(Khan, 2006, Marciano-Cabral, 2003, Visvesvara et al., 2007b).
Research has shown it is possible to build a profile of pathogenicity using a
range of in vitro assays, by assessing physiological characteristics of the amoebae
such as ability: to tolerate higher temperatures and osmolarity; to produce
extracellular proteases (Khan et al., 2000, Sissons et al., 2006); to cause cytotoxic
effects on epithelial cells (Khan et al., 2002, Walochnik et al., 2000); to withstand
human complement (Ferrante and Rowan-Kelly, 1983, Pumidonming et al., 2011,
Toney and Marciano-Cabral, 1998); and show increased sensitivity particularly of
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Swedish GAE study 95
cysts to antimicrobials (Alizadeh et al., 2009, Elder et al., 1994, Lim et al., 2000,
Perrine et al., 1995, Schuster and Visvesvara, 1998).
3.1.1 Aims
To build pathogenicity and molecular profiles of the infecting GAE
Acanthamoeba strains isolated from the Swedish hospital. Strains will be identified
and typed by morphology and genotype techniques, using 18S sequence analysis to
determine T-group and mitochondrial cox1/2 sequence analysis. The pathogenic
potential of the isolates will be analysed by means of an array of in vitro tests,
including trophozoite and cyst sensitivities to a range of antimicrobials. Analysis of
the Swedish hospital samples will determine if the patients are infected with the
same strain of Acanthamoeba, and if that strain is present within the hospital water
system, thereby identifying the source of the infection.
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3.2 Materials and Methods
3.2.1 Establishing amoebae cultures
To establish the presence of FLA within the samples, aliquots were initially
cultured by monoxenic methods (section 2.2.3). All seeded NNA plates were
individually sealed with parafilm, and groups of plates incubated together in plastic
bags, at 30C. Plates were observed at 24-hour intervals to check for growth.
Attempts were made to establish all recovered FLA in axenic culture (section 2.2.4),
occurring over a period of weeks to months.
Amoebae were identified using phase contrast microscopy (inverted)
(Olympus CKX41), with a camera (Olympus, CAMEDIA C5050), following the
dichotomous key for morphological taxonomy (Page, 1988).
Once maintained in axenic culture, strains were cryopreserved for future
work (2.2.5).
All samples were collected from a single ward of Swedish hospital, and
kindly donated by Dr Elisabet Holst, Department of Laboratory Medicine, Lund
University, Lund, Sweden. Patient and hospital strains with as much corresponding
information regarding sample origin as we have access to, is shown in Table 7.
Unfortunately access has not been granted to additional information regarding the
collection of samples, or clinical information.
In addition to Swedish hospital isolates reference strains were included in
the analysis; AK associated A. polyphaga Ros, soil originating A. castellanii (Neff,
50370); GAE associated A. culbertsoni (30171) and A. healyi; and a strain not
associated with disease A. polyphaga (30871).
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3.2.2 Amoebae DNA extraction
Axenic Acanthamoeba cultures were harvested for genomic DNA extraction
by the UNSET method (detailed in section 2.2.6). If attempts to axenise strains had
failed, the cells were then maintained and propagated by the alternative slower
method of monoxenic culturing (section 2.2.3). Acanthamoeba DNA was collected
directly from the agar surface but at lower concentrations than if obtained from
axenic culture. The cells were harvested by repeated washing of the NNA surface
with 8 ml of ice cold dPBS and a pipette. Recovered cells were resuspended in 1 ml
of ice cold dPBS, and subjected to DNA extraction by the UNSET method: Carried
as described in section 2.2.6.1 but with the following modifications: 0.6 ml UNSET
buffer; 0.6 ml phenol-chloroform (1:1); 0.6 ml chloroform:isoamyl alcohol (24:1);
and resuspended in 20 μl of TE0.1 buffer containing RNase A (5 μl/ml).
3.2.2.i Direct DNA extraction from CSF
DNA was extracted directly from CSF samples. Using a technique modified
from the guanidine acetate method to purify target DNA following PCR (section
2.2.10.2).
Added to 25 µl of CSF, were 500 µl of 7 M guanidine acetate and 20 µl of
silica in nH2O (section 2.2.10.2); Guanidine acetate creates a hydrophobic
environment, encouraging the nucleic acids to bind to the silica. The solution was
then heated for five minutes at 55C, mixed briefly by vortex, and incubated on ice
for 10 minutes. The mixture was centrifuged at 10,000 x g for 10 seconds and the
supernatant removed. The DNA bound silica was washed three times in 80%
isopropanol (volumes of 500 µl, 180 µl and 180 µl, respectively), spinning at
10,000 x g for 10 seconds and all supernatant removed between washes. Ensuring
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the eppendorf cap was opened, the DNA bound silica was incubated at ambient
room temperature in a fume hood, for approximately 10 minutes, to allow the silica
pellet to completely air-dry. Finally the DNA was dissolved in 20 µl of pre-warmed
nH2O, and incubated for 5 minutes at 55C (with occasional mixing). The silica was
removed by centrifuging at 10,000 x g for 1 minute, and the supernatant containing
the DNA was recovered in to a fresh eppendorf.
3.2.3 DNA manipulation
Basic manipulation of DNA was carried out with PCR techniques as
described in section 2.2.9, and purification of PCR products as in 2.2.10. Clone
libraries within pGEM®-T Easy cloning vectors were established as described in
2.2.11, production of ultra competent E. coli as in 2.2.12, transformation as in
2.2.13, and purification of propagated plasmids as in 2.2.15. Plasmids were
sequenced commercially as detailed in section 2.2.16. Sequenced results were
analysed and manipulated as described in section 2.2.17. Isolates and clone libraries
were preserved for future use by cryopreservation techniques described in section
2.2.5.
3.2.4 Temperature tolerance assay
The method of Khan and colleagues was used to assess the ability to tolerate
temperature (Khan et al., 2001). This method was carried out by inoculating axenic
Acanthamoeba trophozoites (2.2.4) on to E. coli seeded NNA plates (as described in
2.2.3) and incubating them at 32C and 37C for up to 96 hours. Viability and
growth of the amoebae was determined by microscopy, and identification of the
leading edge of trophozoite migration across the plate surface.
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3.2.5 Osmotolerance assay
The osmotolerance of the amoebae was assayed using a method of Khan and
colleagues to determine pathogenic potential (Khan et al., 2001). The method was
carried out as for temperature tolerance, with the exception of replacing normal
NNA plates (2.2.4) with NNA supplemented with 1 M mannitol (0.25 osmolar).
Plates were incubated at 28C and 37C for up to 96 hours. Growth of the amoebae
was determined by identification of the leading edge of trophozoite migration by
phase contrast microscopy. Mannitol supplemented plates, inoculated with A.
culbertsoni (30171) served as controls.
3.2.6 Protease secretion
A modified method of Maciver was used for extracellular protease secretion
zymography (Edinburgh). This involved sodium dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE) gels containing bovine gelatine (0.1%) (Heussen
and Dowdle, 1980), used to determine the presence of proteases in the
Acanthamoeba culture medium of the strains. Gels were cast containing 2.5 ml
1.5M Tris pH8.8, 4.31 ml dH2O, 100 μl 10% SDS, 44 μl of 22.5% bovine gelatine,
3 ml 30% acrylamide mix 50 μl 10% APS and 20 μl TEMED (modifications from
(Heussen and Dowdle, 1980, Khan et al., 2000)). Acanthamoeba culture medium
(ACM) from pathogenic and non-pathogenic species (5 μl) was mixed with
electrophoresis sample loading buffer (1:1) (2 ml glycerol, 0.5 g SDS, 8 ml 0.5M
Tris, 0.2 ml 0.5% bromephnol blue), and then applied to the gels. Gels were run at
200 V (Mini-PROTEAN®, Tetra Cell, BIO-RAD), in 1X electrode (running) buffer
pH 8.3 (10X: 3.03 g Tris base, 14.1 g glycine, 1 g SDS per 100 ml of dH2O). After
electrophoresis gels were incubated in solution (2.5% Triton X–100, 50mM Tris-
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HCl pH 7.0) for 60 minutes, and then overnight in developing buffer (50mM Tris-
HCl pH 7.0, 2mM CaCl2) at room temperature. Gels were then stained with rocking
for 60 minutes (0.1% Coomassie blue R-250, 40% methanol, 10% acetic acid),
before being rinsed in tap water, and incubated with rocking in destain solution
(10% acetic acid, 5% methanol) for 90 minutes.
Areas of gelatine digestion were visualised as non-staining regions of the
gel. In experimental controls fresh culture media was used, which had not been in
contact with Acanthamoeba isolates.
3.2.7 Complement fixing potential
The method of Pumidonming and colleagues was used to assess the
complement fixing potential of the isolates (Pumidonming et al., 2011). This
method involved harvesting axenic trophozoites by centrifugation at 500 x g for 1
minute. Cells then were washed twice with ¼ strength Ringer’s solution, quantified
using a modified Fuchs Rosenthal haemocytometer, and adjusted to concentrations
of 1 x 104 per ml with ¼ strength Ringer’s solution.
By pooling the sera of four randomly selected healthy-individuals, normal
human serum (NHS) was produced; Whole blood was collected, and allowed to
clot, undisturbed at ambient temperature. All clots were removed by centrifugation
at 1,500 x g for 10 minutes at 8˚C, and the sera removed using a 3ml Pasteur
pipette. All sera were stored at –20˚C, in 0.5ml aliquots, to avoid freeze-thaw cycles
(Invitrogen, 2007).
In a round-bottomed microtitre plate (VWR, Lutterworth, U.K.) 100 μl of
trophozoite suspension was added to 100 μl of NHS. Heat inactivated NHS (56˚C
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for 30 minutes) served as a control (Toney and Marciano-Cabral, 1998). Assay
plates were maintained 28˚C for 1 hour, and the morphology of the trophozoites
following incubation at 0, 5, 10, 20 and 60 minute time points, was examined using
phase contrast microscopy (inverted) (equipped with a Canon EOS 60D camera).
Living trophozoites appeared bright and moving, while killed trophozoites
had destroyed cell membranes. Effect of compliment on the amoebae was also
determined by the formation of a pellet in the bottom of the well. Pellets were only
formed in wells where lysis of the amoebae had occurred, and where healthy
trophozoites remained.
3.2.8 Cytopathogenic potential
A modified method of Khan and colleagues was used to determine the
cytopathic potential of the Acanthamoeba isolates (Khan et al., 2000). The method
involved observing the degradation of epthelioid HeLa cell monolayer’s by
Acanthamoeba trophozoites. Where by HeLa cells were grown in 25 cm2 tissue
culture flasks, in 5 ml Dulbecco’s Modified Eagle Medium (DMEM) (with L-
glutamine) supplemented with 10% FBS (heat inactivated) and incubated in CO2
(5%) at 37˚C, until 80% confluent. Adhered cells were washed in 10 ml dPBS, to
remove all traces of growth media, and detached from the flask surface by the
addition of 2.5 ml of 1X trypsin-EDTA solution and incubation at 32˚C for 5
minutes. Detached cells in suspension were added to 32.5 ml of pre-warmed (37˚C)
DMEM, and 2 ml added to all wells of a 12 well microtitre plate (VWR). Again
incubated in 5% CO2 at 37˚C, until 80-100% confluent.
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Swedish GAE study 102
Acanthamoeba trophozoites were grown as described in 2.2.4, in 5 ml of
semi-defined culture media in 25 cm2 tissue culture flasks, to a confluency of >
80%. Cells were harvested, by centrifugation (500 x g for 1 minute) and washed in
¼ strength Ringer’s solution (three times), before being quantified.
1000 trophozoites in 30 μl were added to each well of the microtitre plate
containing a confluent monolayer of HeLa cell, and incubated in 5% CO2 at 37˚C.
Cytopathogenic effect by the Acanthamoeba was identified as areas of cleared HeLa
cells, and observed by microscopy up to 96 hours post-inoculation. Controls with
epitheloid cells incubated alone but with 30 μl of either ¼ strength Ringer’s solution
or culture medium (without Acanthamoeba) were maintained at the same conditions
for comparisons to be made against.
3.2.9 Antimicrobial sensitivity assays
3.2.9.1 Trophozoites
The methods of Kilvington and colleagues were used for the trophozoite
antimicrobial sensitivity assays (Elder et al., 1994, Hughes and Kilvington, 2001).
This involves trophozoites grown as described in 2.2.4, in 50 ml of culture media in
175 cm2 tissue culture flasks with filter caps, to a confluency of > 80%. Cells were
harvested and washed three times in dPBS-Tween (5% w/v Tween® 80),
centrifuged at 500 x g for 5 minutes between each wash. Finally quantified using a
modified Fuchs Rosenthal haemocytometer, and adjusted to concentrations of 2 x
104 per ml in culture medium.
To test trophozoite sensitivity against antimicrobials, stock solutions of the
compounds, Voriconazole (a gift from Kemprotec Ltd, Maltby, U.K.), Hexamidine
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Swedish GAE study 103
Diisethionate (Désomedire 0.1%- Bausch & Lomb, Kingston upon Thames, U.K.),
Polyhexamethylene biguanide (PHMB) (Cosmosil CQ- Arch U.K. Biocides Ltd,
Castleford, U.K.), Natamycin (Pimaricin- Molekula Ltd, Audenshaw, U.K.) and
Miltefosine were prepared at 1000 μg/ml in nH2O (with the exception of
Miltefosine diluted in 5% ethanol), and filter sterilised (0.2 μm). Antimicrobial
compound dilutions were obtained by two-fold serial dilution of 100 µl with 100 µl
of ¼ strength Ringer’s solution across a flat-bottomed microtitre plate. Calibrated
trophozoites (100 μl) were added to each well, and the plates covered and incubated
in air, at 32˚C, for 48 hours. Compounds were assayed in triplicate, with controls
containing ¼ strength Ringer’s solution only without antimicrobial compounds.
Following incubation, trophozoites were examined by inverted microscopy,
and compared to the controls. Comparison to the test wells allows the degree of
trophozoite growth or destruction to be assessed. The Minimum Trophozoite
Inhibitory Concentration (MTIC) and the Minimum Trophozoite Amoebacidal
Concentration (MTAC) of each test compound against each Acanthamoeba strain
was determined. MTIC is defined as 50% inhibition of trophozoite replication
compared to controls, and MTAC, where all trophozoites are rounded, non-motile
and floating or lysed (Elder et al., 1994, Hughes and Kilvington, 2001).
3.2.9.2 Cysts
The methods of Kilvington and colleagues were also used for the cyst
antimicrobial sensitivity assays (Elder et al., 1994, Hughes and Kilvington, 2001).
This involves Acanthamoeba trophozoites grown as described in 2.2.4, in 50 ml of
semi-defined culture media in 175 cm2 tissue culture flasks with filter caps, to a
confluency of > 80%. From late log-phase cultures, cysts were prepared in Neff’s
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Swedish GAE study 104
constant pH encystment medium (Hughes and Kilvington, 2001, Neff et al., 1964).
Harvested trophozoites were washed in Neff’s encystment medium, and centrifuged
at 1000 x g for 5 minutes, three times. From the pellet approximately 107
trophozoites were added to 100 ml of Neff’s medium in a 175 cm2 tissue culture
flasks with vent/close caps, and left to encyst in a shake incubator revolving at 100
rpm, at 32˚C, for seven days (Hughes and Kilvington, 2001). Following
microscopic evaluation to confirm >90% of cysts were mature; cells were detached
from the flask surface by gentle rubbing of the flask wall with a modified cyst
remover. Made by inserting a sterile polyester tipped applicator (Pur-Wraps)
(Puritan Medical products, Maine, USA) into the upper end (mouthpiece) of sterile
1 ml x 0.01 Sterilin micropipette (Barloworld Scientific Ltd, Stone, U.K.), before
gently heating the micropipette in a Bunsen flame to cause the polystyrene pipette
to soften, and allow the upper end to be manipulated with sterile forceps to have a
90˚ bend. Harvested cells were pelleted by centrifugation at 1000 x g for 5 minutes.
The cyst pellet was then washed three times in ¼ strength Ringer’s solution (2.2.3),
and centrifuged at 1000 x g for 5 minutes. Finally cells were stored in ¼ strength
Ringer’s solution, and stored at 4˚C for use within 14 days.
Cysts were quantified using a modified Fuchs Rosenthal haemocytometer,
and adjusted to concentrations of 2 x 104 per ml in ¼ strength Ringer’s solution.
Antimicrobial cyst assays rely on the adherence of cysts to the bottom of the
polycarbonate wells of the microtitre plates, even after exposure to antimicrobial
compound and removal by washing. Cyst assays were carried out as for
trophozoites, until the end of the 48 hour incubation.
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Swedish GAE study 105
After which adhered cysts were washed three times in ¼ strength Ringer’s
solution with 15 minute soaking between rinses at room temperature. Wells were
then filled with 100 μl of ¼ strength Ringer’s solution containing E. coli JM101
(ATCC 33876) at an O.D.540 of 0.1-0.2, and incubated at 32˚C for 7 days.
Plates were examined for the presence of trophozoites by inverted
microscopy, and the Minimum Cystical Concentration (MCC) was recorded as the
lowest concentration of antimicrobial compound that resulted in no excystment and
trophozoite replication (Elder et al., 1994).
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3.3 Results
3.3.1 Culture and morphological analysis
Diagnosis in suspected cases of GAE is isolation, cultivation and subsequent
microscopy of Acanthamoeba directly from the patient, followed by the sensitive
technique of 18S rDNA sequence analysis.
3.3.1.1 Patient one
Initial samples collected from the patient, tested culture positive for
Hartmannella sp. (Table 7). Hartmannella has yet to be found as the causative
agent of disease in humans, and so given the highly irregular occurrence of
culturing this species from a human sample, additional specimens from the patient
were requested. Of the subsequent samples, four were Acanthamoeba culture
positive (Table 7). The Hartmannella isolated from Patient one, could only be
maintained on seeded-NNA plates.
3.3.1.2 Patient two
The samples included five CSF specimens, collected over a period of five
months, all tested culture positive for Acanthamoeba sp. (Table 7). Axenic culture
was achieved but compared to the hospital water supply samples, growth remained
consistently slower.
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Swedish GAE study 107
3.3.1.3 Patient three
Of the two samples collected from Patient three, both proved culture positive
for Acanthamoeba sp. (Table 7). The earlier of the two specimens collected was a
sample of CSF, followed by a second sample taken from a head wound, two months
later. Again axenic culture was established but Patient three isolates remained
consistently slower when compared to the growth rate achieved by hospital water
supply samples.
3.3.1.4 Hospital water samples
Samples were collected from several areas within the ICU that the patients
had access to, and were associated with water, including a swimming pool, and
several shower rooms. The ICU pool and two shower rooms all tested culture
positive for Acanthamoeba. Axenic culture was established for all isolated strains.
Culture results obtained from this study were relayed back to the hospital,
which responded immediately to super chlorinate the water supply within the
hospital, in an attempt to rid the system of the FLA. Following this treatment,
subsequent samples were collected from the hospital, and subjected to prolonged
repeated analyses, despite continued attempts the sample proved to be culture
negative of FLA.
Repeat testing approximately one year later, identified the presence of
Acanthamoeba (BL2997) (Table 7).
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Swedish GAE study 108
Table 7. FLA isolated from clinical and hospital samples collected in a Swedish
hospital, with morphological identification of genus groups.
Source Strain Origin Culture Identification
Patient 1 a GAE-1 CSF + Hartmannella sp.
GAE- 1a ? - Na
GAE- 1b CSF - Na
GAE- 1c CSF - Na
GAE- 1d CSF - Na
GAE- 1e CSF - Na
GAE- 1f ? + Acanthamoeba sp.
GAE- 1g ? + Acanthamoeba sp.
GAE- 1h ? + Acanthamoeba sp.
GAE- 1i ? + Acanthamoeba sp.
Patient 2 a GAE- 2 CSF + Acanthamoeba sp.
GAE- 2a CSF + Acanthamoeba sp.
GAE- 2b CSF + Acanthamoeba sp.
GAE- 2c CSF + Acanthamoeba sp.
GAE- 2d CSF + Acanthamoeba sp.
Patient 3 a GAE- 3 CSF + Acanthamoeba sp.
GAE- 3a Head wound + Acanthamoeba sp.
ICU Pool a Pool Pool + Acanthamoeba sp.
Shower rooms a SR- 3 Number 3 + Acanthamoeba sp.
SR- 15 Number 15 + Acanthamoeba sp.
Post-super
chlorination a
BL2997 ICU in-water
supply
+ Acanthamoeba sp.
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Swedish GAE study 109
Na: Not applicable; +: Culture positive; -: Culture negative; ?: Source unknown; a:
Donated by Dr E. Holst, Lund University, Lund, Sweden.
3.3.2 Molecular analysis
Despite being culture negative, Acanthamoeba DNA was isolated from a
Patient one’s CSF sample (GAE- 1b). The DNA was directly extracted using a
method with guanidine acetate and silica.
Comparisons made between the PCR findings and those of the culture
methods (Table 8), showed 100% obtainment of 18S and cox1/2 sequence
fragments from all culture positive samples. While from the six culture negative
specimens, one PCR positive result was obtained.
3.3.2.1 18S phylogenetic analysis
A region of 18S rDNA was amplified, cloned and sequenced from all FLA
isolated from the three patients, and the hospital, this included all Acanthamoeba
strains and the single Hartmannella sp. from Patient one. Two 18S primer sets and
corresponding sequencing primers, were used in this study, to reproduce sufficient
sequence length (approximately 204 bp; 1,175-1,379 bp) to determine any variation
for T-group differentiation (Schroeder et al., 2001). All Acanthamoeba 18S rDNA
PCR’s were carried out using the primer pair JDP1F and JDP2R (Schroeder et al.,
2001), producing partial (450 bp) 18S rDNA products. For the Hartmannella sp., a
larger (631 bp) 18S product was obtained with the use of universal primer pair 18S
F & 18S R (Weekers et al., 1994).
Individual BLAST analysis was carried out on all sequences (Altschul et al.,
1997), with nucleotide blast parameters optimised for highly similar sequences
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Swedish GAE study 110
(megablast): Results show the partial 631 bp sequence from Patient one’s
Hartmannella sp. matched by 99% to H. vermiformis (GenBank AY680840
(Kudryavtsev et al., 2005)) .
BLAST analysis of Acanthamoeba sp. isolated from Patient one, Patient two
and Patient three, all matched 100% to Acanthamoeba sp. ATCC 50496 (GenBank
ASU07408 (Gast et al., 1996)). The only Acanthamoeba strain isolated from the
Swedish hospital with an 18S sequence that differed from the others was that of the
ICU pool: Its 18S rDNA sequence matched 100% with A. castellanii ATCC 30010
(GenBank EF554328.1 (Kohsler et al., 2008)).
Analysis of the 18S sequences of all repeat strains isolated from each
patient, confirmed that each was infected with only one strain of Acanthamoeba,
and therefore analysis from this point forwards, was carried out using only one
strain for each patient.
The 18S sequences for Acanthamoeba strains from the three patients and
hospital samples, were aligned to each other using the alignment program for DNA,
ClustalW (2.1) (Larkin M.A. et al., 2007), which allows single nucleotide
differences to be identified. ClustalW identified sequence variation between the two
Acanthamoeba sp. 18S sequence types from this study to be 1.2%: Corresponding to
eight differences in a length of sequence 227 bp long: Six of these were three
double bp omissions from the pool strain, and two were separately occurring base
replacements from cytosine (C) to thymine (T) again in the pool strain.
Using 18S sequences a neighbour-joining distance tree with Kimura two-
parameters correction for multiple substitutions were obtained using MEGA (5.05)
software. Included in the tree are sequences from Acanthamoeba reference strains:
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Swedish GAE study 111
A. castellanii (50373, Neff: type species), A. polyphaga (30871: not associated with
disease) A. polyphaga (Ros: associated with AK), A. healyi (CDC 1283:V013:
associated with GAE) and A. culbertsoni (30171, Lilly: associated with GAE),
along with the Swedish isolates from this study, and a sequence from one of the
closest genera to Acanthamoeba, H. vermiformis (Costa Rica; GenBank AY680840)
(Figure 7).
The tree (Figure 7) confirms the distinction between two strain types within
the Swedish hospital ICU water system: Patient and hospital strains with the
exception of the pool species are identical by 18S analysis; the pool strain differs by
1.5%. This tree confirms only one strain type caused the patients infection. With the
Swedish hospital isolates, from the three patients and the two shower rooms, clearly
cluster away from that of the differing Swedish isolate recovered from the ICU
pool. The pool strain is most similar to A. polyphaga Ros (with 1% dissimilarity).
However, all Swedish strains including the pool isolate belong to the T4 genotype,
based on dissimilarity values of greater than 5%.
The ICU pool strain is found on a branch of its own, supported by a high
bootstrap value of 95: This node clearly separates the T4 isolates (both A.
polyphaga strains and A. castellanii) and the other Swedish hospital strains. Most
species from within the T4 clade in this tree are less than 5% dissimilar from each
other: The only exception is the strain that has not been associated with disease A.
polyphaga (30871), which differs to the pool strain by more than 5% (the cut off
value found within a T-group genotype) (Stothard et al., 1998): However its
dissimilarity it less than the cut off value when compared to soil originating isolate
A. castellanii (Neff, ATCC 50370) and AK associated isolate A. polyphaga Ros.
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Swedish GAE study 112
A maximum parsimony (MP) tree was chosen over one analysed by
neighbour joining algorithms. As it more clearly, by associated bootstrap value
shows a distinction between the T4 strains and those associated with GAE (A.
culbertsoni and A. healyi). Bootstrap values of 99 are found at the node separating
GAE isolates from T4 and Swedish isolates. Unsurprisingly the branch containing
Hartmannella is clearly distinguished from that of Acanthamoeba, and here acts as
a root for the tree.
Patient 1 GAE- 1f
Hospital SR- 15
Hospital BL2997
Patient 3 GAE- 3
Patient 2 GAE- 2
Hospital SR- 3
A. polyphaga Ros
A. polyphaga 30871
A. castellanii 1501/1a
T4
Hospital Pool
T12 A. healyi
T10 A. culbertsoni 30171
Patient 1 Hartmannella sp.
H. vermiformis
88
59
10
8
4
5
13
87
3
95
99
Figure 7. Maximum parsimony distance tree based on partial 18S rDNA sequences
of Swedish hospital FLA with comparison species. The tree is unrooted and
obtained by Kimura two-parameters correction for multiple substitutions using
MEGA (5.05). Analysis is based on reference bp from 1,175 to 1,379. Designated
T-groups and strain origins are shown. Bootstrap values have been included, based
on 1,000 bootstrap values, and placed at the corresponding nodes.
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Swedish GAE study 113
3.3.2.2 Cox1/2 phylogenetic analysis
The BLAST analysis was repeated for the Swedish hospital strains, but this
time using an alternative sequence, that of the mitochondrial cox1/2 gene. Results
follow their 18S sequences, and confirm all Swedish strains except one, match each
other, and in the case of cox1/2, share 89% similarity with A. castellanii (GenBank
U12386.1). Again as with 18S sequence analysis, the differing strain was found to
be that of the ICU pool, which only shared 88% sequence homology with A.
castellanii (GenBank U12386.1).
Sequence variation between the aligned cox1/2 sequences of the ICU pool
strain, and each of those from the three patients and hospital water system, was 4%.
An MP tree was constructed with cox1/2 sequences (Figure 8), and included
the Swedish strains from this study, with the exception of the post-treatment strain
BL2997, and reference strains: A. castellanii (50373, Neff: type species), A.
polyphaga (30871: not associated with disease) A. polyphaga (Ros: associated with
AK), and A. culbertsoni (30171, Lilly: associated with GAE) (Figure 8). As with
18S analysis, two genotypes were represented here from samples collected from the
Swedish hospital ICU. When comparing MP analysis to NJ (Figure 9), the MP tree
contains three nodes with bootstrap values of over 90, while the NJ tree has three
nodes over 75, and one over 90. It is generally considered that bootstrap values of
greater than 70 are evidence to support the distinction of clades. Although the order
of strains is reversed between the two algorithms, the grouping of taxa remains the
same.
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Swedish GAE study 114
Phylogenetic analysis using cox1/2 compared to 18S sequence data provides
more differentiation between the species, where strains are not clumped together
into large genotypes (Figures 8 and 9).
T10 A. culbertsoni 30171
T4 A. castellanii 1501/1a
Hospital Pool
A. polyphaga Ros
A. polyphaga 1501/3aT4
Hospital SR- 15
Patient 1 GAE- 1f
Patient 3 GAE- 3
Patient 2 GAE- 2
Hospital SR- 3
99
97
65
93
16
9
13
Figure 8. Maximum parsimony tree based on partial mitochondrial cox1/2
sequences of Acanthamoeba spp. The tree is unrooted and obtained by Kimura two-
parameters correction for multiple substitutions using MEGA (5.05). The tree is
based on reference bp 8,002 to 8,566. Designated T-groups and origin of strains are
shown. Bootstrap values have been included, based on 1,000 bootstrap values, and
placed at the corresponding nodes.
Patient 3 GAE- 3
Hospital SR- 3
Hospital SR- 15
Patient 2 GAE- 2
Patient 1 GAE- 1f
A. polyphaga Ros
A. polyphaga 1501/3aT4
Hospital Pool
T4 A. castellanii 1501/1a
T10 A. culbertsoni 30171
75
92
78
75
0.02
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Swedish GAE study 115
Figure 9. Maximum parsimony tree based on partial mitochondrial cox1/2
sequences of Acanthamoeba spp. The tree is unrooted and obtained by Kimura two-
parameters correction for multiple substitutions using MEGA (5.05). The tree is
based on reference bp 8,002 to 8,566. Designated T-groups and origin of strains are
shown. Bootstrap values have been included, based on 1,000 bootstrap values, and
placed at the corresponding nodes. The scale bar represents the corrected number of
nucleotide substitutions per base using Kimura method.
3.3.3 Pathogenicity assays
3.3.3.1 Amoebicidal activity of human serum
Normal Human Serum (NHS) exhibited amoebicidal activity against
trophozoites of all seven strains of Acanthamoeba. Observations were made by
microscopy. Following 5 and 10-minute incubation trophozoites appeared live and
viable. After 20 minutes of incubation with the sera, cytopathogenic changes had
begun and trophozoites appeared distressed and rounded. A pellet of killed
Acanthamoeba had formed in all test wells, between 70-80 minutes of incubation
(Figure 10 A-C, and Table 8). Controls were as expected, with trophozoites
incubated for the same period of time in heat-inactivated NHS remaining viable.
A B C
Figure 10. Activity of Normal Human Serum against Acanthamoeba trophozoites.
A, effect of complement from NHS on Acanthamoeba trophozoites; B, control,
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Swedish GAE study 116
Acanthamoeba incubated with heat-inactivated NHS; C, pellet formed in the round
bottomed well after lysis of Acanthamoeba trophozoites.
3.3.3.2 Tolerance of increased temperature and osmolarity
All Acanthamoeba isolates were able to continue trophic growth on NNA E.
coli- seeded plates at both 32C and 37C. However, when challenged in conditions
of high sugar alcohol (1M mannitol), no strains of Acanthamoeba were able to
maintain trophic growth, at either temperature tested. Controls were as expected.
Cysticidal affect of high osmolarity (1M mannitol) was examined, with agar
slices cut from the incubated mannitol plates with trophozoites, and transferred onto
fresh NNA E. coli-seeded plates without mannitol (inverted). Trophic growth of all
strains resumed within 72 hours, and the leading edge was visible emerging from
the upturned slice. Although trophozoites were unable to survive in conditions of
high osmolarity, cysts were formed in the presence of the mannitol, and remained
viable and able to resume trophic growth once conditions became more favourable
(data not shown).
3.3.3.3 Protease secretion assays
Non-stained lanes of the SDS-PAGE confirm areas of gelatine digestion,
and extracellular protease activity. Zymography with ACM has shown all
Acanthamoeba assayed have the capacity to produce proteases (Figure 11). Lanes
associated with the Swedish isolates (1-3, 5 and 6) show a distinction from that of
AK causing A. polyphaga Ros (4). Controls were as expected, confirming proteases
are not present in sterile ACM.
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Figure 11. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-
PAGE) containing gelatine, to observe the presence of extracellular proteases in
Acanthamoeba culture medium (ACM). Lane 1, ICU pool; 2, Shower room 3; 3,
Shower room 15; 4, A. polyphaga Ros; 5, Patient 3; 6, Patient 2; 7, Left blank; 8,
negative control, containing sterile culture media.
3.3.3.4 Cytopathogenicity of Acanthamoeba
In the cytopathogenicity assays, within 96 hours all Acanthamoeba species
had produced varying degrees of cytopathic effect on the HeLa cell monolayer
(Table 8). Of all the strains tested the non-pathogenic A. polyphaga 1501/3a (30871)
and Shower room 3 isolate, equally resulted in the lowest level of` HeLa cell
monolayer degradation. Obvious cleared areas within the monolayer were present,
containing trophic amoebae. Extensive degradation to the monolayer occurred in
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Swedish GAE study 118
wells containing the AK causing strain A. polyphaga Ros, again with numerous
trophic cells. While isolates derived from Patient two, ICU pool, and Shower room
15, all severely destroyed the HeLa cell monolayer with assays of each containing
an abundance of large amoebae full of vacuoles confirming digestion of food
particles. The strain isolated from Patient three caused the most epitheloid
monolayer degradation and resulted in the highest abundance of large digesting
trophozoites. Control assays were maintained for comparison, and both that with
sterile culture media and ¼ strength Ringer’s solution contained an intact fully
confluent HeLa cell monolayer.
Summaries of Acanthamoeba sp. pathogenicity characteristics are shown in
Table 8. Based on results obtained in this study, all strains that were tested are
shown to have at least the potential to be pathogenic. However, because the shower
room 3 isolate and A. polyphaga 1501/3a (30871) exhibited reduced rate of
cytopathic activity towards a monolayer of HeLa cells they could be considered less
pathogenic compared with the known Swedish GAE isolates and AK isolate A.
polyphaga Ros.
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Table 8. Pathogenicity characteristics of Acanthamoeba sp. collected from an
outbreak of GAE with a single ward of a Swedish hospital, and compared with an
AK isolate and a non-pathogen.
Strain Growth Protease
secretion
Cytopathic
activity
Complement
lysis / min
T group
32C 37C 1M mannitol
Patient 2 ●● ●● - Yes +++ < 80 T4
Patient 3 ●● ●● - Yes +++ < 80 T4
Shower
room 3
●● ●● - Yes + < 80 T4
Shower
room 15
●● ●● - Yes +++ < 80 T4
ICU Pool ●● ● - Yes +++ < 80 T4
A.
polyphaga
Ros
●● ●● - Yes ++ < 80 T4
A.
polyphaga
1501/3a
(30871)
●● ●● - Yes + < 80 T4
●●: Good growth; ●: Growth; +: 0-25% of monolayer destroyed; ++: 25-50% of
monolayer destroyed; +++: 50-75% of monolayer destroyed. -: None.
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3.3.4 Drug sensitivity assays
An array of antimicrobial compounds was tested on trophozoites and cysts
of the Acanthamoeba strains (Table 9 and 10).
Against trophozoites, sensitivity assays to determine the Minimum
Trophozoite Amoebicidal Concentration (MTAC) were completed (Table 9).
PHMB exhibited the best antimicrobial activity with average an MTAC of 2.9
μg/ml, followed by miltefosine with an MTAC of 11.7 μg/ml, and hexamidine with
an average MTAC of 23.4 μg/ml. Wide ranges of effective MTAC values were
observed with the therapeutic agents, natamycin and voriconazole. The average
MTAC of natamycin to exhibit antimicrobial activity was 187.5 μg/ml.
Voriconazole showed the highest MTAC, exhibiting antimicrobial activity between
a range of 3.9 - > 125 μg/ml. However the only strain to be killed by voriconazole
within the tested concentration range was AK isolate A. polyphaga Ros, with a
MTAC of 3.9 μg/ml, for all other strains against voriconazole, MTAC values were
greater than 125 μg/ml, and above the range tested (data not shown).
The minimum cysticidal concentration (MCC) was defined as the lowest
concentration of test compound that resulted in no excystment and trophozoite
replication after 48 hours of exposure. Against cysts miltefosine, voriconazole and
natamycin showed little activity, with MCC levels undetected at concentrations
greater than 500 μg/ml. Hexamidine and PHMB were the only therapeutic agents to
show antimicrobial activity towards cysts, within the concentration range tested.
Hexamidine resulted in antimicrobial activity towards 43% of the strains (3/7),
causing a detrimental effect to isolates from Patient 3, MCC level of 62.5 μg/ml, as
well as the pool and shower room 15 isolates, both with MCC levels of 1.9 μg/ml.
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The compound to show the most activity against cysts was the disinfectant PHMB,
with MCC levels of 1.9 μg/ml for 100% of strains (Table 10).
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Table 9. In vitro sensitivities of five antimicrobial compounds against several
strains of Acanthamoeba trophozoites and cysts.
MTAC μg/mla MCC μg/ml
a
Mean Range Mean Range
Miltefosine 11.7 7.8 - 15.6 >500 >500
Voriconazole >125 3.9 - >125 >500 >500
Natamycin 187.5 125 – 250 >500 >500
Hexamidine 23.4 15.6 – 31.25 >500 1.9 - >500
PHMB 2.9 1.9 – 3.9 1.9 1.9
MTAC: Minimum Trophozoite Amoebicidal Concentration; MCC: Minimum
Cysticidal Concentration; PHMB: Polyhexamethylene biguanide; a: n = 7.
Table 10. Acanthamoeba in vitro Minimum Cysticidal Concentrations (MCC),
against five antimicrobial compounds.
Strain MCC μg/ml
Miltefosine Voriconazole Natamycin Hexamidine PHMB
Patient 2 >500 >500 >500 >500 1.9
Patient 3 >500 >500 >500 62.5 1.9
Shower room
3
>500 >500 >500 >500 1.9
Shower room
15
>500 >500 >500 1.9 1.9
ICU Pool >500 >500 >500 1.9 1.9
A. polyphaga >500 >500 >500 >500 1.9
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Ros
A. polyphaga
1501/3a
(30871)
>500 >500 >500 >500 1.9
PHMB: Polyhexamethylene biguanide
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3.4 Discussion
3.4.1 Molecular
Both clinical and hospital samples were analysed for FLA using culturing
methods. Where upon recovery, all amoebae were morphologically classified to
genus level. All recovered Acanthamoeba strains were established axenic culture,
but despite continuous attempts the Hartmannella sp. could not be maintained this
way. Few publications in the literature suggests it is possible to maintain
Hartmannella vermiformis by axenic culture in modified PYNFH medium (Fields et
al., 1990): One group compared several Hartmannella strains in their study but only
H. vermiformis (ATCC 50237), was maintained by axenic techniques, the other two
were cultured in the presence of live, and heat-killed E. coli (Kuiper et al., 2006).
The use of culturing techniques obtained a 73% (16 of 22) recovery of
Acanthamoeba from the samples. In general it is relatively rare to recover viable
Acanthamoeba from CSF samples (Callicott, 1968, Sharma et al., 1993, Singhal et
al., 2001). More often, fluid specimens test PCR positive for Acanthamoeba, when
they have been culture negative. Here, one culture negative sample, tested positive
for Acanthamoeba by PCR using a direct DNA extraction method. With the use of
PCR techniques, 77% (17 of 22) of samples tested Acanthamoeba positive,
highlighting the sensitivity of PCR, and its ability to detect Acanthamoeba where
culturing methods failed. PCR requires the survival of DNA rather than the
numerous intact viable cells that must be present for culturing to be successful.
Although the 18S PCR is a sensitive technique (Ledee et al., 1996) because the gene
is present in such abundance within the cells, for further examination of the strains
and downstream assays such as pathogenicity tests, culturing of the isolates is
required.
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Morphological classification of Acanthamoeba beyond genus level is
extremely difficult and can be subjective. The sensitive nature of PCR and the
ability to use it to classify strains into clades such as T-groups highlights the
valuable of molecular typing, and how it should be used in conjunction with original
culture and microscopic diagnostic techniques.
A previously designed PCR primer pair was used to amplify a highly
variable diagnostic region of the SSU 18S rDNA (204 bp) (Schroeder et al., 2001),
in addition to a pair designed for this study: Which amplifies a 564 bp segment of
the mitochondrial cox1/2 gene, modified from (Kilvington et al., 2004). Both genes
are attractive targets for species differentiation within Acanthamoeba.
The findings from molecular analysis carried out during this study show an
unprecedented case of three patients from the same ward within a Swedish hospital,
all being infected with a single strain of Acanthamoeba: this was confirmed by
identifying all isolates to have identical 18S gene sequences, as well as identical
cox1/2 sequences, by 18S could be typed into T4. A second strain was also
identified from the Swedish hospitals’ water system, originating from the pool: The
isolate was also found to belong to T4, but have different 18S and cox1/2 sequences,
from the others. Until now approximately 150 cases of GAE caused by
Acanthamoeba have been described worldwide, with T4 species as the predominate
group associated with both GAE and AK, followed by T1, T2, T5, T10 and T12
(Lackner et al., 2010, Walochnik et al., 2008).
A related amoeba from the genus Hartmannella was also isolated from the
Swedish hospital sample set, surprisingly originating from a patient. So far
Hartmannella has not reliably been reported as the cause of a fatal disease in
humans (Schuster and Visvesvara, 2004), but early reports within the literature
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often referred to Acanthamoeba as Hartmannella, and used the genus names
interchangeably (Cleland et al., 1982, Jager and Stamm, 1972). Here, Hartmannella
has been associated with human disease, but most likely as an opportunistic
secondary species, as Acanthamoeba was also isolated from the same patient.
The interactions that occur between amoebae are a relatively unexplored
area, but could perhaps begin to explain why Hartmannella has only been found in
humans as a co-infection with Acanthamoeba. In the case of B. mandrillaris, cells
have been found to feed on Acanthamoeba sp. (personal communication, Dr W.
Heaselgrave). While a previous study identified a Hartmannella sp. in association
with a human infection, and determined it was unlikely to be the cause of the
infection, but rather an opportunistic coloniser which may have worsened the
disease (Centeno et al., 1996).
Free-living amoebae are increasingly being recovered from hospital systems,
unsurprising given the ubiquitous nature of Acanthamoeba (Carlesso et al., 2010,
Thomas et al., 2006): Most likely caused by increased frequency of testing and
improvements in techniques, arisen from a better understanding of the need to keep
opportunistic pathogens away from immune compromised hospital patients.
Microbial biodiversity within hospital water systems’ is an area of concern given
the association of FLA with amoeba-resisting bacteria (ARB) and viruses that have
the potential to cause infections in humans (Centeno et al., 1996). To rid a water
system of FLA is almost impossible, amoebae including Hartmannella sp. have
even been isolated from heavily cleaned hospital water systems (Thomas et al.,
2006). The Swedish hospital did however successfully rid their ICU water system of
Acanthamoeba by super chlorinating the system; unfortunately this was only
maintained for a limited period. Retesting approximately 1-year post treatment,
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showed Acanthamoeba of the same genotype and cox1/2 sequence to be present
again.
3.4.2 Pathogenicity
Identification to a genus level is all that is required to diagnose
Acanthamebiasis. However by determining the pathogenicity of the isolate, and its
sensitivity to antimicrobial compounds, the potential to provide substantial benefit
to disease prognosis and the patient’s outcome is gained. Microscopy and
identification based on morphological characteristics alone are not sufficient to
determine the pathogenicity of Acanthamoeba sp.. However, studies have shown a
series of pathogenicity traits can be tested for, to distinguishing pathogenic from
non-pathogenic isolates (Khan et al., 2001, Khan et al., 2002, Khan et al., 2000).
Thermotolerance is an indicator of pathogenicity, (Khan et al., 2001, Khan
et al., 2002, Walochnik et al., 2000), and Acanthamoeba must be able to withstand
the internal temperature of the human body, at 37°C to cause GAE. All the Swedish
isolates were thermotolerant, and grew at 32°C and 37°C, although in the case of the
pool isolate its growth rate was slightly slower by comparison. Additionally both A.
polyphaga species were also thermotolerant, surviving not only 32°C but also 37°C.
Survival at 32°C is not surprising given that A. polyphaga (Ros) is an AK causing
species, originally isolated from a human cornea (Hughes and Kilvington, 2001).
However based on assays completed here, it appears to have the potential to survive
within a human body. Acanthamoeba polyphaga (30871) has previously been
shown to grow at 37°C, further supporting previous evidence to suggest that the
species should be considered pathogenic (Khan et al., 2002, Stothard et al., 1998).
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Osmotolerance is another indicator of pathogenicity, and amoebae that have the
capacity to grow on agar with a higher osmolarity (1M mannitol) are more likely to
be pathogenic and be able to withstand the human body. Here only A. culbertsoni
(30171) the control, exhibited osmotolerance and grew on mannitol-containing
plates. Although all the Swedish isolates were able to form viable cysts that could
resume trophic growth once osmotic pressure had been removed. Previously,
evidence has shown A. polyphaga (30871) to exhibit osmotolerance (Khan et al.,
2002), yet here the strain was unable to grow on mannitol-containing plates. When
considering osmolarity in relation to pathogenicity characteristics, epidemiological
information for given isolates must be taken into consideration. Osmotolerant A.
griffini (1501/4) was classified as a weak pathogen, despite never having been
associated with disease, its ability to withstand increased osmotic environments is
more likely to attributed to having originated from the sea around the USA coast
(Khan et al., 2001). The exact reasons why these strains did not grow on mannitol-
NNA plates is unclear, but despite this all other tests suggest they were pathogenic.
To cause infection, Acanthamoeba have to infiltrate the human body’s
defence system. In ocular infections amoebae must cross the stromal layer of the
cornea, and the blood-brain barrier in encephalitis. Very little is known about the
exact mechanism of how the pathogenic cascade is achieved, but for invasion to
take place, interactions between the amoebae and host cells must occur, most likely
involving the host extracellular matrix (da Rocha-Azevedo et al., 2010). It has been
shown that secreted amoebic proteases have the ability to degrade components of
the host extracellular matrix, such as collagens I, III, and IV, elastin, fibronectin and
laminin (He et al., 1990, Hurt et al., 2003, Na et al., 2001, Sissons et al., 2006). By
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inhibiting proteases, the capabilities of Acanthamoeba to invade collagen and BD
Matrigel (basement membrane matrix) are reduced (da Rocha-Azevedo et al.,
2010). Here all isolates including the Swedish water strains produced extracellular
proteases. The pathogenesis of Acanthamoeba infections is highly complex and
multifaceted involving proteolytic activity. Proteases are degradative enzymes that
are vital for migration and tissue invasion. All Acanthamoeba isolates tested to
date, including pathogenic and non-pathogenic produce extracellular proteases, to
catalyse the hydrolysis of large proteins into smaller molecules ready for absorption
by the cell (Khan, 2009).
Acanthamoeba are free-living organisms, which happen to be opportunistic
pathogens with a secondary ability of being able to cause infection. As all isolates
tested here produce extracellular proteases, it was not unexpected to observe that
they all too exhibit cytopathic effects on epitheloid cells when incubated together.
However, subtle differences were found between some of the strains, when
compared to each other. The length of time taken to reach the same point of
destruction varied amongst the strains: With isolates from shower room 3, and A.
polyphaga (30871) causing destruction to the HeLa cell monolayer at a slow rate,
which was only marginally slower than the AK isolate A. polyphaga (Ros).
The innate resistance factor complement is the host’s powerful first line of
defence against invading Acanthamoeba. Although many microorganisms are
susceptible to complement lysis, some are better suited to withstand its lytic activity
than others (Jokiranta et al., 1995), which includes the highly pathogenic species A.
culbertsoni (Toney and Marciano-Cabral, 1998). Resistance to complement by
some Acanthamoeba strains seems to show some correlation to pathogenicity,
although reports have shown both pathogenic and non-pathogenic strains are
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susceptible to lysis by complement (Ferrante, 1991, Ferrante and Rowan-Kelly,
1983, Pumidonming et al., 2011).
Ultimately in vitro analyses have shown no single Swedish hospital strain to
be any greatly more virulent than any of the others, and in fact they have
comparable pathogenicity traits to A. polyphaga Ros and A. polyphaga 30871. For
an Acanthamoeba infection to take hold, multiple virulence factors must be met.
Which accounts for the rarity of the disease despite the ubiquitous nature of
Acanthamoeba within the environment. Never before has an outbreak of GAE been
documented, suggesting extraordinary circumstances had occurred within the
Swedish hospital involving and/or the amoebae or patients. Pathogenicity assays
have confirmed that all the Swedish hospital isolates are virulent but perhaps the
line between pathogenic and non-pathogenic is not so clearly defined, and because
all strains have the ability to produce extracellular proteases (Khan, 2009) for
example, they also have an innate capacity to cause disease, providing predisposing
factors allow.
To provide a fuller picture of virulence, several other pathogenicity assays
could have been included, such as longevity tests observing cyst endurance in
storage over an extended period of time. As well as alternative extracellular
proteases assays, identifying their presence: Invasion/migration assays can establish
if the trophozoites have the ability to pass through a layer of gelatine collagen, or
BD Matrigel (basement membrane matrix) (da Rocha-Azevedo et al., 2010, Hurt et
al., 2003) to a lower chamber containing nutrients. Proteases can be classified into
six major groups including, serine, aspartic, cysteine, metalloproteases, threonine,
and glutamic acid (Khan, 2009), determination of the type and also quantity
produced by each strain, will improve the understanding its pathogenicity potential.
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A full examination of the Swedish hospital system should also be carried out,
identifying if the system supports large numbers/types of microorganisms making it
an ideal home to harbour pathogenic FLA.
3.4.3 Antimicrobial sensitivity
There is no current effective treatment for GAE or disseminated
acanthamebiasis, and very few people have ever responded favourably to therapy.
For antimicrobials to be successful in treating GAE, they like the amoebae must be
able to cross the blood-brain barrier, and enter the brain and CSF. Most
acanthamoeba infections, are treated with antimicrobial combinations, and of those,
success has been achieved with intravenous pentamidine isethionate in combination
with topical chlorhexidine and ketoconazole administered to a transplant patient
who contracted disseminated infection (Slater et al., 1994): An HIV/AIDS patient,
received fluconazole and sulfadiazine combined with surgical excision of the brain
lesion (Seijo Martinez et al., 2000): While a previously healthy paediatric, received
ketoconazole and had a total excision of the infected area (Ofori-Kwakye et al.,
1986): Combination therapy with oral miltefosine and amikacin were used to
successfully treat a patient with GAE in Austria (Walochnik et al., 2008). Another
successful outcome arose from a 17-year-old immunocompromised patient who
presented with acute purulent meningoencephalitis, and was treated with a
combination of meropenem, linezolid, moxifloacin, and fluconazole (Lackner et al.,
2010). The strategy of combining antimicrobial compounds may result in beneficial
synergistic effects, and additionally avoid any potential resistance patterns.
Particularly as in vitro sensitivity testing of clinical isolates results in strain and
species differences, highlighting that no single compound can be assumed effective
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against all amoebae (Schuster and Visvesvara, 1998). Further compounded by the
poor correlation observed between in vitro MCC findings and patient response to
those therapeutic agents (Elder et al., 1994, Kilvington et al., 2002).
Unfortunately as access was not granted to any patient clinical information from
the Swedish outbreak, sensitivity assays were designed using a range of
antimicrobial compounds, with the intention of hopefully testing the strains in vitro
against a compound that they were exposed to during the course of treatment: If not
the actual compound, at least one belonging to the same family.
For this study in vitro cyst sensitivity testing, identified all strains could resist
cidal effects of miltefosine, voriconazole and natamycin at concentrations lower
than 500 μg/ml. Hexamidine also showed little effect towards cysts, at
concentrations lower than 500 μg/ml for four of the strains (Patient 2, Shower room
3, A. polyphaga Ros, and A. polyphaga 30871): Whilst being cysticidal to Patient
3’s isolate at concentrations above 62.5 μg/ml, and to Shower room 15 and the Pool
isolates at greater than 1.9 μg/ml. However good cysticidal activity against all
strains was detected in assays with PHMB (>1.9 μg/ml).
Polyhexamethylene biguanide (PHMB) is an antiseptic in the same family as
chlorhexidine but is not as cytotoxic. It is used to good effect as a treatment of AK,
where it is administered as a topical solution of 0.02%, either alone or in
combination therapy. As it has a low mammalian toxicity, it can therefore be
applied over long periods of time. Its structure is a positively charged polymer, with
low surface tension, that against Acanthamoeba targets the plasma membrane by
binding with the acidic phospholipids. As PHMB is a disinfectant it is only suitable
for topical application, and despite having good cysticidal effect on many strains of
Acanthamoeba it cannot be used to treat systemic infections. Several AK cases have
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been documented, reporting no response to treatment with PHMB, even though
isolated amoebae were found to be sensitive to the compound in in vitro assays
(Elder et al., 1994, Murdoch et al., 1998, Perez-Santonja et al., 2003). Clearly
inconsistencies between in vitro sensitivity profiles and management of individual
cases of acanthamebiasis can occur. In vitro drug sensitivity assays are undoubtedly
important in the development of potential new compounds to treat acanthamoeba
infections, but such assays clearly have limitations: Can any effects occurring
within the carefully controlled environment of a microtitre well fully represent the
human body? Effects of the antimicrobials on the amoebae may be altered by host
cellular and humoral responses, potentially affecting the course of the disease.
This study reported some cysticidal effect caused by the antimicrobial agent,
hexamidine di-isethionate (Desomedine): The compound is a homologue of
propamidine but has been shown in some cases to have greater cysticidal activity
(Brasseur et al., 1994, Perrine et al., 1995), and disputed more recently (Seal, 2003).
The diamidine group, contains many homologues including not only propamidine
but, butamidine, heptamidine, hexamidine, octamidine, pentamidine and
nonamidine (Perrine et al., 1995). The antimicrobial properties of the diamidines’
against Acanthamoeba have been found to be proportional to the length of the alkyl
chain of the diamidine molecule, linked to its lipophilicity and ability to penetrate
the amoeba cell membrane. Which causes structural changes to the plasma
membrane leading to alterations of cell permeability (Perrine et al., 1995). Currently
the recommended treatment regime for AK includes an aromatic diamidine, either
hexamidine (Desomedine) or propamidine isethionate (Brolene) at 0.1% (1 mg/ml),
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in combination with a disinfecting biguanide either 0.02% PHMB or chlorohexidine
digluconate (Khan, 2009).
Here, hexamidine against cysts showed variable amoebicidal results: with very
weak cytotoxic effects against four strains >500 μg/ml; limited towards another at
62.5 μg/ml, with strong activity shown against two strains, at 1.9 μg/ml. Variability
was also shown against trophozoites, with an average MTAC of 23.4 μg/ml, with a
range of 15.6 – 31.25 μg/ml. Such variability in the effectiveness of one agent
reflects the suggestion that natural variation in Acanthamoeba species accounts for
much of the variation found in sensitivity assays to determine cidal effects caused
by antimicrobials (Elder et al., 1994, Khan, 2009).
In all cases by in vitro assays, trophozoites were more susceptible to
therapeutic compounds than cysts, as has been previously reported (Elder et al.,
1994, Jones et al., 1975, Walochnik et al., 2009). As with cysts assays, PHMB
showed the most activity against strains, with average MTAC values of 1.9 µg/ml.
Next best amoebicidal activity towards the strains was shown by the promising
compound Miltefosine (Impavido®, hexadecylphosphochlorine): An
alkylphosphocholine anticancer agent that appears to hold necessary therapeutic
properties for the treatment of AK (Walochnik et al., 2009), and GAE (Walochnik
et al., 2008). Although the mode of action is unknown, miltefosine appears to
accumulate in the cell membrane causing structural disruption and affecting cell
metabolism (Eibl and Unger, 1990, Walochnik et al., 2009). Recently it has been
used for the treatment of disseminated Acanthamoeba infections, and is also a
potential antimicrobial agent for treatment of AK and GAE (Mrva et al., 2011).
Miltefosine has been shown to be effective against clinical and environmental
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isolates of Acanthamoeba, starting at concentrations of 2 µg/ml (~5 μM) (Schuster
et al., 2006a, Walochnik et al., 2002), and 25.5 μg/ml (62.5 μM) (Mrva, M 2011):
Here assays showed amoebicidal effects at 11.7 μg/ml, between the strong and weak
amoebicidal activity previously shown.
Natamycin (Natacyn®, 5% (50 mg/ml)) is a macrolide polyene antifungal
agent used to treat fungal eye infections. The macrolide antibiotic group is large,
containing many agents including amphotericin B, erythromycin, azithromycin and
clarithromycin. The macrolides have broad-spectrum activities against a range of
microorganisms (Mattana et al., 2004), and have been used to treat GAE, with both
successful outcomes (Nachega et al., 2005, Walia et al., 2007), and failure (Kuashal
et al., 2008). The precise mode of action of the macrolides within Acanthamoeba is
not fully understood but thought to involve the plasma membrane and ergosterol
(Raederstorff and Rohmer, 1985), altering cell permeability (Mattana et al., 2004).
Antifungal agents, natamycin (>500 μg/ml), and voriconazole (>500 μg/ml)
showed only weak amoebicidal effects against cysts, but higher cytotoxicity against
trophozoites was attained, natamycin (187.5 μg/ml) and voriconazole (>125 μg/ml).
Voriconazole belongs to the azole group, and includes the homolog compounds,
clotrimazole, miconazole, ketoconazole, and fluconazole. It has a strong inhibitory
effect upon Acanthamoeba trophozoites but not cysts (Schuster et al., 2006a), with a
mode of action that is similar to that of natamycin (Sanati et al., 1997).
Through the use of in vitro sensitivity assays, several pharmaceutical agents
have been shown to have antimicrobial properties towards Acanthamoeba cysts and
trophozoites, but no single compound has proven effective against all isolates of the
genus: Reflecting the high natural variability within the species. This only goes to
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highlight the importance of carrying out drug sensitivity tests for every case of AK
and GAE diagnosed. Which would not only help identify the best choice compound,
but also help discount any that are potentially ineffective. The Amoebae Laboratory,
at the University of Leicester now routinely carries out such sensitivity testing for
the Leicester Royal Infirmary, and other UK hospitals. Their experience has shown
clinical strains of AK usually show sensitivity to biguanides, but sensitivity to all
other drug compounds in particular diamidines, is very variable (personal
communication Dr W. Heaselgrave). So it is important to identify the effective
treatments quickly to improve patient prognosis. However, cases that fail to respond
to medical therapy do occur despite the apparent in vitro sensitivity to compounds
such as PHMB (Elder et al., 1994, Murdoch et al., 1998, Perez-Santonja et al.,
2003). The cause of this is unknown, but for these patients prognosis is poor, as they
remain culture positive and have to undergo repeated penetrating keratoplasty. With
Acanthamoeba infections, prompt instigation of treatment is necessary for good
prognosis. Therefore sensitivity testing is important to ensure therapy stands a
chance of being effective and is not a waste of time.
3.4.1 Conclusion
Epidemiological implications and a better understanding of the course of
infection may occur as a result of speciation of the isolate involved in
acanthamebiasis. The value of the sequencing systems has been clearly shown here,
by allowing strain differentiation. Severely debilitated patients on a paediatric ICU,
within a Swedish hospital fell ill with symptoms of GAE. Clinical samples were
collected from the three patients and any water-associated places that the patients
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had access to. Epidemiological typing was carried out, with CSF samples cultured
for FLA, confirming the patients were all infected with T4 Acanthamoeba isolates.
In addition to the Acanthamoeba, one patient had also contracted an extremely
rare associated infection with a species of Hartmannella as well.
The combined use of two molecular genotyping systems both 18S and cox1/2,
confirmed the infection had originated from the water system within the hospital
and the patients were all infected with the same strain. The T-group and cox1/2
systems both proved to be independently rapid, sensitive and highly effective tools
for use within epidemiological studies.
Pathogenicity and antimicrobial sensitivity assays confirmed the isolates
originating from the Swedish hospital were all virulent, but no more pathogenic
than expected given that they were associated with an unprecedented outbreak of
GAE.
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References 138
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